key: cord-012089-haqqrwad authors: baez, antonino; cho, kwang-myung; liao, james c. title: high-flux isobutanol production using engineered escherichia coli: a bioreactor study with in situ product removal date: 2011-03-10 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3173-y sha: doc_id: 12089 cord_uid: haqqrwad promising approaches to produce higher alcohols, e.g., isobutanol, using escherichia coli have been developed with successful results. here, we translated the isobutanol process from shake flasks to a 1-l bioreactor in order to characterize three e. coli strains. with in situ isobutanol removal from the bioreactor using gas stripping, the engineered e. coli strain (jcl260) produced more than 50 g/l in 72 h. in addition, the isobutanol production by the parental strain (jcl16) and the high isobutanol-tolerant mutant (sa481) were compared with jcl260. interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either jcl16 or jcl260. this result suggests that in situ product removal can properly overcome isobutanol toxicity in e. coli cultures. the isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by clostridium in a similar integrated system. the growing need for alternative energy calls for new strategies to produce liquid fuels such as alcohols, alkanes, and fatty acid esters (or biodiesel) from renewable sources (dellomonaco et al. 2010; peralta-yahya and keasling 2010; solomon 2010) . compared to ethanol, higher alcohols (e.g., 1-propanol, isobutanol, n-butanol, 2methyl-1-butanol, 3-methyl-1-butanol) have higher energy density and lower hygroscopicity, which make them better candidates as gasoline additives or substitutes. however, no native organisms have been identified to produce these advanced biofuels in appreciable quantities (clomburg and gonzalez 2010) . for example, isobutanol was identified as a by-product in beer fermentation, but with a titer as low as 16 mg/l (garcia et al. 1994) . since the feasibility of a bioprocess depends critically on the final product titer (stephanopoulos 2007) , a synthetic approach to produce higher alcohols from nonfermentative pathways in escherichia coli was previously devised (atsumi et al. 2008b ). in addition, metabolic engineering efforts to produce these alcohols at higher titers and yields have been performed (atsumi et al. 2008a, b; cann and liao 2008; clomburg and gonzalez 2010; connor and liao 2008; jarboe et al. 2010; shen and liao 2008; smith et al. 2010) . this strategy uses the amino acid biosynthetic pathway and diverts its 2ketoisovalerate for isobutanol synthesis by overexpression of 2-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adha). because of the ubiquity of the amino acid pathways, this strategy is compatible with many organisms and shows significant promise for industrial applications. in particular, atsumi et al. (2008b) engineered an e. coli strain (jcl260) as a host for isobutanol production. genes that code for proteins involved in by-product formation from pyruvate and acetyl-coa were deleted in jcl260 (table 1) with an intention to increase pyruvate availability for isobutanol synthesis. such deletions improved the isobutanol production of jcl260 strain compared to its parental strain, jcl16, under micro-aerobic conditions in shake flasks (atsumi et al. 2008b) . even without any process optimization, atsumi et al. (2008b) were able to produce 22 g/l of isobutanol in 112 h with a yield of 86% of the theoretical maximum. although this titer is 85% and 12% higher than those reported by ezeji et al. (2003) and qureshi and blaschek (1999) , respectively, for n-butanol produced by clostridium in batch bioreactors, industrial-scale fermentation processes require higher final titers to be economically feasible. the final product titer can be limited by various factors depending on the particular production strain and bioprocess. since isobutanol is toxic to the cell, isobutanol production from glucose may be limited by the toxicity of the final product itself. in this sense, improving the tolerance of the biocatalyst becomes a primary necessity to achieve a process with high product titers (alper et al. 2006; gonzalez et al. 2003; jarboe et al. 2010; miller and ingram 2007; yomano et al. 1998 ). it has been reported that e. coli was unable to grow at isobutanol concentrations >8 g/l (brynildsen and liao 2009) . even though the e. coli strain continued to produce isobutanol after it ceased to grow (atsumi et al. 2008b) , product toxicity may eventually damage the cell and limit the final titer (rutherford et al. 2010) . in this regard, atsumi et al. (2010b) isolated and characterized an isobutanol-tolerant e. coli strain (sa481) able to grow at 8 g/l. sa481 was evolved from the high isobutanol producer strain (jcl260) by a sequential transfer method increasing isobutanol concentration every 15 transfers. the sa481 strain showed superior growth characteristics compared to the high producer (jcl260) strain when they both were cultivated at 6 and 8 g/l of isobutanol. compared with the control condition, specific growth rate for jcl260 decreased by 35% and 74% at 6 and 8 g/l of isobutanol (calculated from data showed by atsumi et al. 2010b) , respectively, while sa481 growth was not affected at 6 g/l of isobutanol and decreased just 13% at 8 g/l. interestingly, atsumi et al. (2010b) also showed that sa481 did not produce more isobutanol than jcl260 under normal conditions. however, the experiments were performed in shake flasks. accordingly, the performance of sa481 harboring psa65/psa69 for isobutanol production in bioreactors will be evaluated in this work. the other approach to solve the cytotoxicity problem and its detrimental effect on the final titer from a bioprocess perspective is the in situ product removal. several in situ solvent recovery strategies have been developed for n-butanol fermentation (groot et al. 1992; nielsen and prather 2009; roffler et al. 1987; qureshi et al. 2005) . since gas stripping is a simple and efficient way to recover the solvent from the fermentation broth (inokuma et al. 2010; lee et al. 2008) , in the present work, gas stripping integrated with fermentation was used to compare the isobutanol production by the high producer (jcl260), isobutanol-tolerant (sa481), and the parental (jcl16) strains carrying psa65 and psa69 plasmids. we hypothesize that if isobutanol could be removed from the fermentation broth, the production can be extended significantly and the final titer will surpass the 22 g/l obtained previously in flask cultures. strains, plasmids, and pre-culture strains and plasmids used are shown in table 1 . a single colony of freshly transformed cells was inoculated into a 250-ml screw-cap flask containing 25 ml of luria-bertani media with antibiotics (0.05 g/l kanamycin and 0.1 g/l ampicillin). overnight pre-culture was performed at 37°c on a rotary shaker (250 rpm). bioreactor culture conditions cultures of e. coli were performed in 1-l stirred tank bioreactor (applikon biotechnology, schiedam, the netherlands) using a working volume of 0.35 l. the bioreactor was inoculated with 2% of overnight preculture and the cells grown at 37°c. after 2 h, 0.1 mm iptg was added and the temperature was decreased to 30°c to start isobutanol production. the induction time represents the beginning of fermentation described in fig. 2 . isobutanol production was tested at two temperatures, 30°c and 37°c, for jcl260. the ph was controlled at 6.8 by automatic addition of 2 m naoh solution. dissolved oxygen (do) was maintained above 20% with respect to air saturation by raising stirrer speed (from 200 to 600 rpm). air was bubbled in the bioreactor with two goals: (1) provide oxygen and (2) in situ isobutanol stripping out. air flow rate was maintained at 0.5 vvm the first 3 h of isobutanol production and then was increased to 1.2 vvm in order to remove isobutanol from the culture broth. the evaporated isobutanol was condensed using two graham condensers connected in series ( fig. 1 ). the exhaust gases from the fermentor were bubbled in a trap (picker b containing 800 ml of water; fig. 1 ) cooled with ice and then circulated through condenser 1 (l 300 mm and cooling coil surface area 400 cm 2 ; fig. 1 ) maintained at 4°c. after that, gas continued circulating through a second equal loop (d receiver and condenser 2; fig. 1 ). fermentation was allowed to proceed in batch mode until entry into the stationary phase (between 9 and 10 h), and then an intermittent manual feeding was started to avoid glucose depletion. fermentation samples were collected to determinate growth, isobutanol production, and organic acid and glucose concentrations. cell growth was followed by optical density measurements at 600 nm and converted to dry cell weight concentration by calibration plots of samples dried to constant weight in an oven at 80°c. glucose concentration was determined with ysi biochemical analyzer (ysi instruments, yellow springs, oh, usa). isobutanol concentration was quantified by a gas chromatograph (gc) model 6850 (agilent technologies, santa clara, ca, usa) equipped with flame ionization detector 6850 series (agilent technologies). a 30-m, 0.32-mm i.d., 0.25-μm db-ffap capillary column was used. gc oven temperature was initially held at 85°c for 3 min and raised with a gradient 45°c/min until 225°c and held for 3 min. helium at 1.7 ml/min was used as the carrier gas with 9.52-psi inlet pressure. a volume of 1 μl supernatant of the culture broth was injected using 1-pentanol as internal standard. acetate concentration was determined by high-pressure liquid chromatography (agilent technologies) with an aminex hpx-87h column (bio-rad laboratories, hercules, ca, usa) maintained at 35°c. a mobile phase of 5 mm h 2 so 4 was used at 0.6 ml/min. cells were harvested by centrifugation at 10,000×g for 10 min at 4°c from bioreactor cultures, washed twice with 50 mm potassium phosphate buffer (ph 7.0), and stored frozen at −80°c. frozen cells were suspended in the same washing buffer, blended with glass beads, and disrupted in series 4 × 1 min. crude extract was centrifuged at 10,000×g at 4°c for 30 min and the supernatant used for enzyme assays. the concentration of the protein preparation was determined by the bradford method. acetolactate synthase (alss) and dihydroxy-acid dehydratase (ilvd) assays were performed as described by atsumi et al. in the present work, bioreactor cultures of jcl260 were performed by controlling do above 20%, ph at 6.8, and temperature at 30°c and were compared with jcl16 and sa481. a high aeration rate (1.2 vvm) was used for in situ isobutanol recovery. typical kinetics of isobutanol produc-tion, cell growth, glucose consumption, and acetate production are shown in fig. 2 . isobutanol concentration plotted in fig. 2a represents the total isobutanol produced by the culture quantified from receivers b, d (fig. 1) , and fermentation broth. the total isobutanol concentration was calculated as a sum of isobutanol quantities determined in receivers b, d, and broth culture considering a working volume of 0.35 l. note that we condensed isobutanol into water again for easy quantitation. one could directly condense isobutanol without redissolving into water. isobutanol production continued even after cell growth was stopped (fig. 2a, c) . in fact, around 80% of total isobutanol accumulated by jcl260 cultivated at 30°c was produced after the growth phase. such behavior was similar to that reported by atsumi et al. (2008b) , but 2.3-fold higher. the high isobutanol producer strain (jcl260) accumulated 50.8±1.1 g/l of isobutanol in 72 h and the parental (jcl16) 41.4±4.5 g/l in the same time (fig. 2a) . surprisingly, the high isobutanol-tolerant strain (sa481) produced only 23±4.4 g/l of total isobutanol in 68 h. after 22 h, the maximum isobutanol concentration in the fermentation broth for jcl260 and jcl16 was around 11 g/l and for sa481 around 6.5 g/l (fig. 2b) . the maximum cell concentrations for jcl16, sa481, and jcl260 were 8.4±0.3, 7.6±0.7, and 6.7±0.16 g/l, respectively (fig. 2c) . since the aeration rate, temperature, and pressure (hence the desorption rate) were maintained constant and at the fig. 3 volumetric isobutanol production rate calculated as slope from time profile of isobutanol production before and after 22 h for all cultures, except for jcl260 at 37°c where it was determined before and after 16 h. error bars represent the difference between duplicate cultures fig. 4 time profile of acetate production by jcl260 poxb − mutant and high isobutanol producer (jcl260) strains harboring psa65/psa69 plasmids cultivated in shake flasks containing 25 ml of medium. data shown are the results of three independent fermentations same level for all cultures, the differences in the isobutanol concentration accumulated in the fermentation broth can be attributed to differences in the isobutanol production rate (figs. 2b and 3) . isobutanol production rate did not remain constant and decreased progressively after the growth phase. as inferred from fig. 2b , the isobutanol production rate decreased dramatically after 22 h, for jcl260 and jcl16 cultivated at 30°c, suggesting that some events (e.g., loss of enzyme activities or substrate depletion) took place at that time. accordingly, volumetric isobutanol production rate was calculated before and after 22 h of culture, being higher before 22 h (fig. 3) . jcl260 and jcl16 reached the maximum cell density around 10 h and sa481 at 22 h. the cell density of sa481 was constant until the end of culture, while jcl260 and jcl16 presented a slight decrease (fig. 2c) , presumably due to the differences in isobutanol tolerance. the cell growth stopped around 10 h (jcl260 and jcl16) and cells did not grow even after feeding. initial glucose concentration was approximately 55 g/l and maintained above 2 g/l in all cultures by intermittent additions of 500 g/l glucose solution (fig. 2d) . since isobutanol production mainly comes from glucose carbon and not from yeast extract (atsumi et al. 2008b) , glucose depletion was avoided because long starvation times (more than 1 h) diminished the isobutanol production (data not shown). completely aerobic conditions were maintained in all cultures, and lactate, formate, and ethanol were not detected in the broth culture. acetate was the major by-product detected, which is a well-known result of carbon imbalance during overfeeding, known as overflow metabolism (farmer and liao 1997; wolfe 2005; wong et al. 2008) . maximum acetate concentrations reached during the growth phase (first 10 h; fig. 2e ) were 4.8±0.8, 7.5±2.6, and 3.5±0.5 g/l for jcl260, jcl16, and sa481, respectively. jcl16 and jcl260 accumulated 13.7±2.6 and 7.1±3.6 g/l of acetate at 22 h, respectively. although jcl260 accumulated less acetate than parental (jcl16), pta deletion in jcl260 was not enough to avoid acetate accumulation (fig. 2e) . since the strain still had the poxb path to acetate production, its contribution was tested by deleting the poxb gene (fig. 4) . thus, jcl260 and jcl260 δpoxb mutant were compared in flask cultures. isobutanol production and maximum cell density were similar in both strains (fig. 4a, b) . glucose consumption for jcl260 was just slightly higher than jcl260 δpoxb. however, acetate accumulated by jcl260 at 16.5 h was twofold higher than the δpoxb strain. this result suggested that poxb did contribute to the acetate accumulation. additionally, both strains were also tested in bioreactor cultures with integrated stripping out system, and the results are shown in fig. 5 . growth and isobutanol time profile were different for jcl260 and jcl260 δpoxb mutant (fig. 5a-c) . unexpectedly, jcl260 δpoxb did not grow as well as jcl260 and produced less isobutanol, while acetate accumulation was similar for both strains. deletion of pta and poxb (jcl260 δpoxb strain) did not fig. 5 time profile of acetate production by jcl260 poxb − mutant and high isobutanol producer (jcl260) carrying psa65/psa69 in bioreactor cultures. data shown are duplicated and single fermentation for jcl260 and jcl260 poxb − , respectively eliminate acetate accumulation, but even a triple mutant (δack δpta δpoxb) was not enough to eliminate acetate accumulation during aerobic fermentation (chang et al. 1999; phue et al. 2010) , suggesting that acetate might also come from other pathways. time profile of high isobutanol producer strain (jcl260) at 30°c and 37°c since vapor pressure increases with temperature, higher temperature should increase the gas stripping rate, and consequently, isobutanol concentration in the fermentation broth should be lower. accordingly, the isobutanol production by jcl260 was evaluated at 37°c and compared with the production at 30°c (fig. 2a) . unfortunately, the maximum isobutanol concentration was twofold lower at 37°c compared with that at 30°c. the isobutanol concentration in the fermentation broth reached 5.3 g/l and decreased until 0.4 g/l at the end of culture (fig. 2b) . the maximum biomass concentration (7.2 g/l) was practically the same for both temperatures (fig. 2c) . glucose depletion was avoided by intermittent feeding in all cultures (fig. 2d) . acetate producfig. 6 enzyme activity of isobutanol pathway for jcl260 carrying psa65/psa69 plasmids in bioreactor cultures at 30°c (open symbols) and 37°c (closed symbols). a total isobutanol production calculated as sum of isobutanol concentrations from receivers b, d (fig. 1) , and broth culture considering a working volume of 0.35 l. b time profile of glucose concentration and cell growth. c acetolactate synthase (alss) activity. d dihydroxy-acid dehydratase activity. e 2-ketoisovalerate decarboxylase (kivd) activity. f alcohol dehydrogenase (adha) activity. error bars indicate the difference between duplicate assays tion was higher at 37°c (fig. 2e) , reaching a maximum concentration of 15.8±1.6 g/l at 10.5 h. initially, it was hypothesized that the lower isobutanol production at 37°c was due to the poor quality of heterologous protein expression. to test this hypothesis, activities of four (alss, ilvd, kivd, and adha) enzymes involved in the biosynthetic isobutanol pathway were determined in bioreactor cultures performed at 30°c and 37°c (fig. 6) . isobutanol, growth, and glucose time profile are shown in fig. 6a , b, and in concordance with the results of fig. 2a , maximum isobutanol concentration reached at 30°c was twofold higher than at 37°c. enzyme activities of alss, ilvd, and kivd were roughly similar in both 30°c and 37°c cultures (fig. 6c-e) . however, the adha activity (fig. 6f) in the 37°c culture was significantly lower than that in the 30°c culture from 17 h to the end of cultures, suggesting that this step may contribute to the difference between the two temperatures. kinetic and stoichiometric comparison of the strains the specific growth rate (μ), volumetric productivity, and isobutanol yield on glucose were determined (fig. 7) . the specific growth rate for jcl260 was similar to the parental strain (jcl16) and was 5% lower for sa480. as expected, the specific growth rate for jcl260 at 37°c was higher (around 26%) than at 30°c. in agreement, the acetate production was higher at 37°c than at 30°c because the acetate production rate is directly related to the specific growth rate (eiteman and altman 2006) . the volumetric productivity of jcl260 at 30°c was 0.7 gl −1 h −1 , 93% higher than the productivity of clostridium beijerinckii ba101 for 1-butanol production estimated from data reported by ezeji et al. (2003) using fermentation cultures with similar feeding and in situ product removal strategies (gas stripping integrated with fermentation process). in contrast, the volumetric productivity for jcl16 and the isobutanol-tolerant strain (sa481) was lower than jcl260. the maximum isobutanol yield on glucose (determined at the end of fermentation) for jcl260 cultivated at 30°c was 0.29±0.003 g/g, which is 68% of the theoretical maximum. a lower isobutanol yield on glucose was obtained for jcl260 at 37°c and for sa481. batch fermentations were integrated with gas stripping to evaluate the isobutanol production of the highproducing (jcl260), high isobutanol-tolerant (sa481), and parental (jcl16) strains. for the first time, we were able to produce more than 50 g/l of isobutanol in 72 h. we found that with in situ product removal, e. coli was able to surpass the 22 g/l produced in flask culture (atsumi et al. 2008b) . although sa481 has shown superior isobutanol tolerance to jcl260 (atsumi et al. 2010b) , it was not reflected in the final titer of isobutanol obtained in the integrated process. these results suggest that under these culture conditions, isobutanol production was not limited by growth or the product toxicity during growth phase. atsumi et al. (2010b) found that isobutanol production, cell growth, and glucose consumption rate of sa481 tested in flask cultures were similar to those of jcl260 and that improved tolerance of sa481 did not increase the final titer. in this study, glucose consumption of sa481 was lower than that of jcl260; consequently, isobutanol production rate was also lower (figs. 2a and 3) . cell growth of sa481 stopped around 22 h, reaching practically the same cellular concentration as jcl260. at this time, isobutanol concentration in the broth was around 7 g/l and cells did not grow even though there was no apparent nutrient limitation. on the other hand, temperature should be an important factor in isobutanol production since the final titer at 37°c was 53% lower than at 30°c. the observed difference might be caused by a lower adh activity at 37°c toward the end of the culture (fig. 6e) in addition, it has been reported that a shift-up in growth temperature decreases the proportion of unsaturated fatty acids in the membrane (balamurugan 2010; casadei et al. 2002; ingram and buttke 1984) . such changes in membrane composition may determine the tolerance level of cells to a particular stress. for example, ingram and buttke (1984) observed that alcohol tolerance of zymomonas mobilis reduces with increasing growth temperatures. if such behavior will be the same for e. coli, poor isobutanol production at 37°c could be justified by a reduced alcohol tolerance compared with 30°c. fig. 7 comparison of specific growth rate (μ, liters per hour), volumetric productivity (grams per liter per hour) and product yield on glucose (y p/s , gram per gram) of the high isobutanol producer (jcl260), high isobutanol-tolerant (sa481), and parental (jcl16) strains cultured at 30°c and jcl260 at 37°c. the μ was calculated during the exponential growth phase. error bars indicate the difference between replicate cultures brynildsen and liao (2009) reported that 8 g/l (1%) of isobutanol caused a growth arrest in e. coli, but even a lower concentration of solvent (0.8%, v/v, or 6.4 g/l of nbutanol) caused a 50% growth decrease, disruption in respiratory efficiency, increased permeability of the cell wall, and oxidative stress response on e. coli (rutherford et al. 2010) . in this work, cell growth of jcl260 and jcl16 stopped around 10 h (when isobutanol concentration in the broth was roughly 7 g/l) and cells did not grow even after feeding. such a phenomenon might be attributed to solvent toxicity because isobutanol concentration at 10 h was similar to those tested by brynildsen and liao (2009) and atsumi et al. 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van den bosch, p. l. f.; abbas, b.; janssen, a. j. h.; muyzer, g. title: microbiological analysis of the population of extremely haloalkaliphilic sulfur-oxidizing bacteria dominating in lab-scale sulfide-removing bioreactors date: 2008-10-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1598-8 sha: doc_id: 12431 cord_uid: l2i5utne thiopaq biotechnology for partial sulfide oxidation to elemental sulfur is an efficient way to remove h(2)s from biogases. however, its application for high-pressure natural gas desulfurization needs upgrading. particularly, an increase in alkalinity of the scrubbing liquid is required. therefore, the feasibility of sulfide oxidation into elemental sulfur under oxygen limitation was tested at extremely haloalkaline conditions in lab-scale bioreactors using mix sediments from hypersaline soda lakes as inoculum. the microbiological analysis, both culture dependent and independent, of the successfully operating bioreactors revealed a domination of obligately chemolithoautotrophic and extremely haloalkaliphilic sulfur-oxidizing bacteria belonging to the genus thioalkalivibrio. two subgroups were recognized among the isolates. the subgroup enriched from the reactors operating at ph 10 clustered with thioalkalivibrio jannaschii–thioalkalivibrio versutus core group of the genus thioalkalivibrio. another subgroup, obtained mostly with sulfide as substrate and at lower ph, belonged to the cluster of facultatively alkaliphilic thioalkalivibrio halophilus. overall, the results clearly indicate a large potential of the genus thiolalkalivibrio to efficiently oxidize sulfide at extremely haloalkaline conditions, which makes it suitable for application in the natural gas desulfurization. electronic supplementary material: the online version of this article (doi:10.1007/s00253-008-1598-8) contains supplementary material, which is available to authorized users. the presence of h 2 s in fuel gases causes many environmental and technical problems demanding its removal before combustion. usually, it is done by a catalytic oxidation. an alternative bioprocess based on lithoautotrophic sulfide-oxidizing bacteria (sob) as a catalyst has been developed and successfully applied at full scale in the netherlands for biogas desulfurization (buisman et al. 1990; janssen et al. 1995 janssen et al. , 1999 . the major principle of the thiopaq technology is regulation of sulfide oxidation at the level of elemental sulfur by low redox potential, which provides two advantages over the complete oxidation to sulfate: (1) the oxidation does not generate protons but regenerates hydroxyl ions, thereby allowing to save on caustic absorbent; (2) formation of elemental sulfur allows easy separation of the final oxidation product and recirculation of the liquid phase. in case of biogas, the bioprocess is performed at relatively low salt concentrations and ph, i.e., at 0.5 m total na + and ph 8.2-8.5, with bicarbonate as the dominant anion in solution. at these conditions, marinetype sob, such as halothiobacillus neapolitanus w5 (visser 1997) , function very well. however, for the removal of h 2 s from high-pressure natural gas and sour gas streams produced in the petrochemical industry, the total alkalinity must be substantially increased to make scrubbing process more efficient. this dictates a shift in a type of biocatalyst from neutrophilic marine sob to natronophilic (sodaphilic), highly salt tolerant sob (banciu et al. 2004a; van den bosch et al. 2007 van den bosch et al. , 2008 . such sob have recently been discovered in the sediments of hypersaline soda lakes (sorokin and kuenen 2005; sorokin et al. 2006a ). one out of three genera of haloalkaliphilic sob described so far, the genus thioalkalivibrio, is characterized by the ability to grow in saturated soda brines containing up to 4 m total na + and ph from 7 to 10.5. all but one out of nine described species of this genus are obligatory alkaliphilic and soda-philic; that is, they can only grow in carbonate brines and at a ph above 8. a single species, thioalkalivibrio halophilus, is a facultative halophile capable of growth at neutral ph in nacl brines as well as in soda brines at ph 10 (banciu et al. 2004b ). all previously described high salttolerant thioalkalivibrio strains were obtained from the enrichments with thiosulfate as substrate and at high redox potential, since batch cultivation at low redox potential with sulfide as an electron donor is much more complicated. therefore, such organisms might be unsuitable for application in the thiopaq process module, where the redox potential is as low as −350 mv and sulfide/polysulfide is the actual electron donor. this paper describes results of a microbiological investigation of lab-scale bioreactors oxidizing sulfide/polysilfide at ph 8.5-10 and a salt content of 2-3 m na + /k + . it is shown that the populations were dominated by extremely salt-tolerant alkaliphilic sob, represented by two subgroups of the genus thioalkalivibrio. surface sediment samples (0-10 cm) were obtained from hypersaline soda lakes in northeastern mongolia, southwestern siberia, and wadi al natrun in egypt. eight to 12 samples from individual lakes in each region were com-bined into a single pool. the ph of the brines varied from 9.2 to 10.6, total salt concentration from 60 to 400 g l −1 , and total soluble alkalinity from 0.05 to 3 m. two types of lab-scale bioreactors were used for oxidation of sulfide at oxygen-limited conditions. one was a 5-l gaslift column (fbr) fed by h 2 s gas where oxygen supply was controlled by a redox electrode (vs ag/agcl; van den bosch et al. 2007 van den bosch et al. , 2008 . another type was a 1-l stirred tank reactor (sl-br) where sodium sulfide was fed in sequential fed-batch mode (1 mm shots) and oxygen was supplied by limited diffusion through a loop from silicon tubing ( supplementary fig. 1 ). the major difference between these two types of bioreactors was the concentration of polysulfide. in the fbr, it was generally maintained at a relatively low level of 100 μm sulfane using redox control. the fraction of free sulfide as compared to polysulfide was minimal at ph 10 and increased toward ph decrease. in the sl-br, the concentration of polysulfide reached 1 mm right after addition of sulfide due to a rapid spontaneous reaction with sulfur formed during the previous stage, decreasing gradually to zero due to biological activity of the sob. therefore, the latter conditions were selective for high polysulfide/sulfide resistance and polysulfide as a substrate. the duplicate fbr were run during 2 years at variable modes at ph starting from 10.1 and ending at 8.8 (table 1) . the redox potential was maintained most of the time below −390 mv (vs ag/agcl). the mineral medium was based on bicarbonate/carbonate buffer containing 70% k + and 30% na + , 2 m in total. the presence of so much potassium is certainly unusual and never occurs in natural soda lakes. the reason behind employing such an unusual buffer is the much higher solubility of potassium carbonates as compared to sodium carbonates, especially when ph is decreased below 9 and bicarbonate is the dominant anion. our preliminary tests with extremely haloalkaliphilic cultures of the genus thioalkalivibrio demonstrated that many strains can withstand up to 50% replacement of na + by k + at 2 m total cation in the form of carbonates, and a few strains even tolerated 90% replacement. this is in contrast to the neutrophilic halophilic sob, which cannot grow already at 30% replacement of nacl by kcl (sorokin 2008) . the n source in the bioreactors was urea. a more detailed description of these bioreactors is given elsewhere (van den bosch et al. 2007 (van den bosch et al. , 2008 . the duplicate 1-l sl-brs were run within 2 months at 1.2-3.0 m of k/na carbonates, 70% k, and ph 10.1. the n source was ammonia. isolation and cultivation of pure cultures of extremely haloalkaliphilic sob two approaches were used for the enrichment and isolation of pure cultures of haloalkaliphilic sob. one was based on using thiosulfate as substrate (20 mm), which is stable at aerobic conditions. alternatively, sulfide (2 mm) was used as a substrate. use of sulfide is much more difficult because of its volatility, toxicity, and spontaneous oxidation. the former effects, however, are reduced at highly alkaline ph, while the spontaneous oxidation was reduced by a ten times decrease in trace metal content and the use of only 1% oxygen in the gas phase. to standardize the enrichment procedure, the same conditions were also used in the thiosulfate enrichments. the liquid mineral medium used for enrichment and isolation contained carbonate buffer with 2 m k/na (70% k), ph 10, 1 g/l of k 2 hpo 4 , and 4 mm of either nh 4 cl or urea. after sterilization, the medium was supplemented with trace metals (pfennig and lippert 1966) , 1 mm mgcl 2 , and a sulfur substrate. serial dilutions were incubated at 30°c in 20-ml hungate tubes with 2 ml medium and a headspace containing 1% o 2 in argon. when turbidity appeared in a highest dilution, it was plated into a solid agar medium of the same composition. the plates were incubated in closed jars (3 l) under the atmosphere of argon containing 1% o 2 . in case of sulfide enrichments, the plates did not contain any substrates. instead, 4 ml of 1 m sodium sulfide solution was placed into the jar in a 10-ml vial. during the prolonged incubation (2-4 weeks), h 2 s entered the gas phase and was absorbed by the alkaline agar creating optimal conditions for sulfide-utilizing sob. dominating colony types were picked under the binocular and placed into the liquid medium. the purity of the culture was checked by repeated plating. the activity and the stoichiometry of oxidation of various sulfur compounds was studied with an oxygen electrode using washed cells as described previously (banciu et al. 2004b ). the ph dependence was examined at 2 m total na + , using the following buffers: for ph 6-8, 0.1 m 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid and nacl; for ph 8-11, a mixture of sodium bicarbonate/sodium carbonate containing 0.1 m nacl. to study the influence of salt concentration, sodium carbonate buffer with ph 10 containing 0.1 and 4.0 m of total na + was applied. all buffers contained 50 mm k + and 1 mm mg 2+ . cell membranes were obtained by ultracentrifugation of the sonified cells at 144,000×g for 2 h (beckman). the membranes were resuspended in soda buffer at ph 9 containing 0.5 m total na + and used to measure several enzyme activities. cytochrome c oxidase was measured by the rate of oxidation of 1 mm n,n,n′,n′-tetramethyl p-phenylenediamine (tmpd) spectrophotometrically at 610 nm. sulfide-quinon reductase (sqr) activity was determined in a discontinuous assay of anaerobic decyl-ubiquinondependent oxidation of sulfide (0.2 mm each). cytochrome c content in the membranes was estimated from spectroscopic measurements of dithionite-reduced minus air-oxidized preparations using uv-visible diod-array spectrophotometer (vectra 8453, hp, amsterdam) and molar absorbance coefficient e 550 20 mm cm −1 . chemical analysis of sulfur (sulfide, sulfur, thiosulfate, and sulfite) and nitrogen (nitrite and ammonium) compounds and cell protein were performed as described previously (sorokin et al. 2001; banciu et al. 2004b) . sulfane sulfur atoms of polysulfide were analyzed in the same way as free sulfide, i.e., after precipitation as zns. zero-valent sulfur in polysulfide was analyzed in the same way as free sulfur (i.e., by cyanolysis of acetone extract) after decomposition of polysulfide molecules by acid treatment. total protein comparison of the isolates and the reactor biomass was performed using 5-15% gradient sodium dodecyl sulfate polyarcylamide gel electrophoresis (sds-page) according to laemmli (1970) . repetitive-sequence-based polymerase chain reaction (rep-pcr) fingerprinting comparison of the sob isolates was performed with the gtg5 primer set as described previously (foti et al. 2006 ). genomic deoxyribonucleic acid (dna) was extracted from the cell pellet using the ultraclean soil dna extraction kit (mobio laboratories, usa), following the manufacturer's instructions. for the pure cultures, the nearly complete 16s ribosomal ribonucleic acid (rrna) gene was obtained using general bacterial primers gm3f (5′-agagtttgatcctggctcag-3′) and gm4r (5′-tacγγttac-cttgttacgactt-3′). for the denaturing gradient gel electrophoresis (dgge) analysis, partial amplification with a primer pair 341f+gc/907r was employed (schäfer and muyzer 2001) . dgge was performed as described by muyzer et al. (1993) , using a denaturing gradient of 20-35% to 60-70% denaturants in 8% polyacrylamide gel. individual bands were excised, reamplified, and run again on a denaturing gradient gel to check their purity. pcr products for sequencing were purified using the qiaquick pcr purification kit (qiagen, the netherlands). the sequences were first compared with sequences stored in genbank using the blast algorithm (http://www.ncbi.nlm.nih.gov/blast). subsequently, the sequences were imported into the arb software program (ludwig et al. 2004) , automatically aligned, and added to a phylogenetic tree using the quick-add tool. subtrees were then built using the neighbor-joining algorithm with automatic selected correction settings. respiration profiles for different sulfur compounds were determined with washed cells directly from the bioreactors and used to characterize the activity and ph/salt response of the dominant sob populations. a general trend could be seen that sulfide and, in case of the sl-br reactors, polysulfide were much better respiratory substrates for the mixed sob population in the reactors than thiosulfate (fig. 1a) . this is not usually the case with the sob dominating fully aerobic conditions (sorokin et al. 2006a ). sulfide/polysulfide specialization indicates that there should be a different pathway for oxidation of these highly reduced electron donors as compared to thiosulfate oxidation. one of the specialized components of such a pathway might be sulfide-quinone reductasea flavin-containing enzyme oxidizing sulfide to elemental sulfur with quinones as electron acceptors (griesbeck et al. 2000) . we found a relatively high activity of this enzyme in the biomass of the analyzed bioreactors (fig. 1b) . another parameter characteristic for a highly active respiratory chain in aerobic chemolithoautotrophs is the activity of cytochrome c oxidase together with the presence of a high-potential cytochrome c pool. this pair is responsible for the terminal delivery of the electrons obtained during oxidation of electron donor to oxygen. comparison of the three reactor samples demonstrated that only in fbr2 (ph 8.8), it was at a level normal for aerobic haloalkaliphilic sob (personal data on pure cultures), while in sl-br2 (ph 10) and fbr1 (ph 9.5), the cytochrome c oxidase had a low specific activity. this might be connected to the condition of extremely low redox potential in the reactors, especially in sl-br2, due to the presence of polysulfide at a high concentration. one of the very important parameters of the sob biomass activity in the reactors is its ph response. comparison of the two fbr reactors, run at different ph (9.5 for fbr1 and 8.8 for fbr2), demonstrated a different ph response (fig. 2) . there was a clear difference in the neutral part of the ph profiles for sulfide between the two reactors. this indicates either the presence of a separate phneutral population in fbr2 (ph 8.8) or a dominance of facultatively alkaliphilic sulfide-oxidizing species in this reactor. the profile for polysulfide in fbr2 was similar to that of sulfide. in contrast, the profile for thiosulfate oxidation by fbr2 cells was typical for obligate alkaliphilic sob. the latter profile might be due to the presence of different sob populations specialized either in sulfide/ polysulfide or thiosulfate or due to a different ph response of the same population with different substrates. our pure culture data favor the first suggestion. but, in general, the respiratory data clearly indicated domination of haloalkaliphilic sob in the reactors. a direct comparison of the membrane proteins, which are dominated by the respiratory enzymes, showed major similarity between the two reactors fbr1 and fbr2 (fig. 3) , suggesting a presence of the same dominant sob population. molecular analysis of the biomass from two sl-br and two fbr bioreactors based on 16s rrna gene dgge showed low genetic diversity, typical for autotrophic mix cultures with domination of one to two sob genotypes and some side heterotrophic populations (fig. 4) . since analysis of the sl-br and fbr biomasses was performed on different gels, the profiles could not be directly compared. fig. 1 metabolic activity of sob biomass from sulfide-oxidizing bioreactors (see table 1 ). a respiratory activity of whole cells with different sulfur substrates at ph 10 and 2 m total na/k (carbonates). however, a general similarity of the fbr1 and fbr2 (ph difference 0.7 units) profiles were quite obvious, while the profiles of two sl-br (difference in salinity is equivalent to 2 m k/na) looked different. this might be explained by the fact that while ph 9.5 and 8.8 are still within alkaliphilic range, a salt content of 1.5 and 3 m total na/k usually selects for different groups of haloalkaliphilic sob (sorokin et al. 2006a) . the phylogenetic analysis of the dgge band sequences demonstrated that in all samples, lithoautotrophic sob belonging to the gamma-proteobacteria were dominant (fig. 5) , although its affiliation differed at different reactor conditions. at moderate haloalkaline conditions (reactor sl-br1), the dominant band belonged to a distant relative of the genus halothiobacillus, while at higher salt (2-3 m k/na), both in sl-br2 and in fbr, the representatives of the genus thioalkalivibrio were identi(banciu et al. 2004b ). other identified sequences either belonged to nonsulfur-oxidizing alkaliphilic heterotrophs, such as marinospirillum (sl-br2) or bacillus (fbr2), or to haloalkaliphilic facultative sulfur oxidizers, for which sulfide oxidation might provide additional energy (sorokin et al. 2000 (sorokin et al. , 2006b , such as alkalispirillum (fbr1) and rhobacteraceae (fbr2). the cultivation approach demonstrated the presence of culturable sob in the reactor biomass, mostly at a very high density (table 1) . from the highest positive dilution, more than 20 pure cultures were obtained either with thiosulfate or with sulfide as the sulfur substrate. all but one of them (strain alr 15) were obligately chemolithoautotrophic sob identified as the representatives of other undescribed strains, presented in the tree, were isolated from various soda lake sediments: akl11, akl15, and tvmix90, from kulunda steppe (siberia, russia); almg14, from mongolia; ale20 and aln 1, from wadi natrun (egypt); alj24, from lake magadi (kenya). scale bar represents 1% sequence divergence three subclusters within the genus thioalkalivibrio by 16s rrna gene sequencing (fig. 5) . most of the isolates from the sl-br reactors (ph 10) and from the fbr run at ph 10 were clustering around the core group of the genus which includes the type species thioalkalivibrio versutus and its close relatives thioalkalivibrio thiocyanoxidans, thioalkalivibrio jannaschii, and thioalkalivibrio nitratis, all obligate haloalkaliphiles. only two isolates, alr 3 and alr 6, obtained from the high-ph fbr on sulfide, belonged to the cluster of facultative alkaliphilic halophile t. halophilus. on the other hand, all the isolates obtained with sulfide as substrate from the reactor with lowest ph (fbr2, ph 8.8) belonged to the t. halophilus subcluster, and only those enriched with thiosulfate clustered with the core group of obligate alkaliphiles. the latter results were consistent with the dgge analysis (figs. 4 and 5) of the biomass from fbr2 and with the ph profile of respiration (fig. 2) . on the other hand, the cultivation-based approach failed to produce the same results for sl-br2, where molecular data also identified a dominance of a t. halophilus-like population, while only representatives of the core group of the genus thioalkalivibrio were obtained in culture. this might be explained by the different culture conditions: in batch enrichment, cultivation at ph 10 from the sl-br2 might have provided better conditions for the secondary population of obligate alkaliphiles. a ph-salt response of one of the t. halophilus-like isolates, strain alr 14, was studied in more details, since this type seemed to be an important player in both types of bioreactors. growth experiments with thiosulfate demonstrated that, despite being phylogenetically closely related to facultatively alkaliphilic t. halophilus, strain alr 14 can be regarded as an obligate alkaliphile (fig. 6a) . on the other hand, its respiratory activity (fig. 6b) , especially with sulfide, was quite high already at subalkaline ph values (8.5-9.0), indicating good fitness to the reactor conditions (ph 8.8). salt profiles corresponded to those of extremely salt-tolerant moderate halophiles (fig. 6c) and also fit very well to the reactor conditions (2 m total na/k). an interesting ph effect was observed in case of the oxidation of sulfide and polysulfide by alr 14 (fig. 6d) : the oxidation of sulfane atoms (s − ) proceeded mostly to elemental sulfur at near neutral ph, while its further oxidation to sulfate was increasing at an alkaline ph range. a similar effect of ph on product formation was found in the fbr bioreactors (van den bosch et al. 2008) , with one important difference: in case of washed cells of a pure culture, the final oxidation product was sulfate, while in the bioreactor, thiosulfate accumulated at high ph. this difference can be accounted to the nature of sulfane atom oxidation in the two systems. in the bioreactors, enzymatic oxidation of polysulfide sulfane atoms was apparently inhibited by very low redox potentials, resulting in spontaneous oxidation to thiosulfate. in contrast, the washed cells experiment was conducted at a high initial oxygen concentration, allowing enzymatic conversion of sulfane with sulfate as the final product. a single heterotrophic isolate was obtained from fbr2 from the special colonies formed on alkaline plates incubated under the h 2 s-containing gas phase. since also dgge analysis indicated a presence of heterotrophs in the reactor, there must be a source of organic carbon in the system. such carbon could be provided by a dominant population of thioalkalivibrio either by excretion or after lysis of dead cells. usually, sob are forming sulfur inside the colonies. in contrast, strain alr 15 produced large sulfur halos around the colonies (supplementary fig. 2) . further investigation showed that the bacterium, which was identified as a member of the genus halomonas, was an obligate heterotroph unable to actually use sulfide or thiosulfate as an energy source. however, during heterotrophic growth, it oxidized thiosulfate to tetrathionate, which is a well-known property of this group of gamma-proteobacteria (sorokin 2003) . our scenario to explain this extracolonial sulfur formation is the following (supplementary fig. 2 ): h 2 s was being absorbed into the alkaline agar from the gas phase and partially converted to elemental sulfur. the latter reacted with sulfide to form polysulfide (indeed a yellowish coloration was observed after prolonged incubation of the plates). spontaneous reaction of polysulfide with oxygen resulted in the formation of thiosulfate. in this reaction, the active role of the bacterium starts, in providing tetrathionate-a powerful oxidant for sulfide. the reaction of sulfide with tetrathionate produces sulfur and regenerates the substrate (thiosulfate) for the heterotroph. so, only traces of thiosulfate are necessary to catalyze the oxidation of sulfide to sulfur in the presence of tetrathionate-forming heterotrophs. such a microbiochemical catalysis has been demonstrated for a marine heterotrophic bacterium catenococcus (sorokin et al. 1996) . the difference with halomonas alr 15 is in the internal generation of thiosulfate from polysulfide, which is stable only at highly alkaline conditions. in the case of catenococcus, thiosulfate had to be supplied externally. although the colonies with external sulfur accumulation were quite common in our enrichments, this organism was not found in the reactor biomass by molecular analysis, which makes its role in sulfide oxidation in situ in comparison with the obligate autotrophic thioalkalivibrio questionable. our previous microbiology investigation of various soda lakes demonstrated a presence of highly diverse population of the obligately lithoautotrophic sob of the genus thioalkalivibrio which was dominating especially at ex-treme salinity (sorokin, kuenen 2005; sorokin et al. 2006a; foti et al. 2006) . so, it is not surprising that in the bioreactors inoculated with the sediments from hypersaline soda lakes, these haloalkaliphilic sob species were dominating. the reactor isolates were very closely related to the thioalkalivibrio species obtained from the natural sediments isolated at fully oxic conditions with thiosulfate as substrate. this might mean that both low-potential oxidation of sulfide/polysulfide and high-potential thiosulfate oxidation pathway may coexist in the same sob species. indeed, that's what usually can be seen in the respiratory profiles of the haloalkaliphilic sob grown either with thiosulfate or sulfide at fully aerobic conditions, i.e., equal rates of sulfide-and thiosulfate-dependent respiration. on the other hand, the respiratory tests with the sob biomass from the low redox potential bioreactors clearly indicated sulfide/polysulfide preference (see fig. 1 ), suggesting a possibility of a different pathway for oxidation of these highly reduced electron donors as compared to thiosulfate oxidation. one of the specialized components of such a pathway might be sulfide-quinone reductase-a flavin-containing enzyme oxidizing sulfide to elemental sulfur with quinones as electron acceptors (griesbeck et al. 2000) , which activity was detected in the reactor biomass. furthermore, the profound "sulfide/polysulfide specialization" of the biomass in sl-br2 and fbr1 correlated with a relatively low cytochrome c oxidase activity. taken together, this might indicate a substantial change in the electron flow pathway and needs further, more detailed investigation. another interesting result of this study is a diversity of a single culturable haloalkaliphilic sob taxon found in the bioreactors at triple extreme conditions (high alkalinity, high total salt, high k). although all of them belonged to a single genus, most of the isolates were genetically different from each other. the exact meaning of such difference is not completely clear, although some phenotypic difference, such as correlation with the ph in the reactor and with the substrate, used for isolation, can be seen in different subgroups. apparently, the genus thioalkalivibrio, despite being a relatively narrow specialized ecotype, possesses extraordinary potential for microadaptation. on the other hand, the molecular analysis (dgge) of the reactor populations indicated a presence of a single thioalkalivibrio phylotype closely related to a facultatively alkaliphilic species t. halophilus. it is not easy to explain such a big difference between the results from two approaches. we can offer two. first, this could be a result of cultivation bias, since batch enrichment/isolation from the sl-br reactors was performed at ph 10 and resulted in isolation of numerous strains of obligate alkaliphiles related to the core group of the genus thioalkalivibrio. perhaps, if lower ph values were used, isolates related to t. halophilus would be obtained at least with sulfide as the substrate, similar to the results with fbr reactors. a second explanation might be that the dna from the obligately alkaliphilic types of thioalkalivibrio, for one or another reason, was less accessable for pcr-a usual bias of the dgge method. judging from the previous results of the microbiological study of natural alkaline lakes, the major factor determining a domination of these two types of thioalkalivibrio is ph in combination with high chloride content. for example, t. halophilus-like sob-dominated among the isolates from the haloalkaline wadi natrun lakes in egypt with nacl as a dominant salt and ph around 9, while natrono (soda)philic thioalkalivibrio species dominated in true soda lakes with ph around 10 where sodium carbonates were present at molar concentration (sorokin et al. 2006a ). the genus thioalkalivibrio has a great potential to thrive at extremely haloalkaline conditions and is a sure candidate for application in biological sulfide removal directly from spent sulfide caustics. in the natural soda lake sediments, often another genus of low salt-tolerant alkaliphilic sob (thioalkalimicrobium) can be found, which is characterized by extremely high rates of sulfide oxidation (sorokin and kuenen 2005; sorokin et al. 2006a ). the fact that it apparently was not present in the described bioreactors could probably be explained by high salt concentration which is above its capacity to grow. application of haloalkaliphilic sulfur-oxidizing bacteria for the removal of h 2 s from gas streams thioalkalivibrio halophilus sp. nov, a novel obligately chemolithoautotrophic facultatively alkaliphilic and extremely salt-tolerant sulfur-oxidizing bacterium from a hypersaline alkaline lake optimization of sulphur production in a biotechnological sulphideremoving reactor genetic diversity and biogeography of haloalkaliphilic sulfur-oxidizing bacteria belonging the genus thioalkalivibrio biological sulfide oxidation: sulfide-quinone reductase (sqr), the primary reaction biological sulfide oxidation in a fed-batch reactor removal of hydrogen sulfide from wastewater and waste gases by biological conversion to elemental sulfur. colloidal and interfacial aspects of biologically produced sulfur particles cleavage of structural proteins during the assembly of the head of bacteriophage t4 arb: a software environment for sequence data profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna über das vitamin b 12 -bedürfnis phototropher schwefel bacterien denaturing gradient gel electrophoresis in marine microbial ecology diversity of halophilic sulfur-oxidizing bacteria in hypersaline habitats haloalkaliphilic sulfur-oxidizing bacteria in soda lakes denitrification at extremely alkaline conditions in obligately autotrophic alkaliphilic sulfuroxidizing bacterium thioalkalivibrio denitrificans sulfur cycling in catenococcus thiocyclus roseinatronobacter thiooxidans gen. nov., sp.nov.-a new alkaliphilic aerobic bacteriochlorophyll a-containing bacterium from soda lake. microbiology haloalkaliphilic sulfur-oxidizing bacteria increased metabolic versatility of haloalkaliphilic bacteria belonging to the alkalispirillum-alkalilimnicola group from soda lakes sulfide oxidation at halo-alkaline conditions in a fedbatch bioreactor the effect of ph on thiosulfate formation in a biotechnological process for the removal of hydrogen sulfide from gas streams sulfur compound oxidation and sulfur production by thiobacillus sp. w5 (now-halothiobacillus) acknowledgements this work was supported by stw (wbc.5939) and nwo-rfbr (47.011.2004.010) with a financial contribution from shell intertnational and paque b.v. (the netherlands).open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012085-ubdzhkfq authors: jin, tao; zhang, tong; ye, lin; lee, on on; wong, yue him; qian, pei yuan title: diversity and quantity of ammonia-oxidizing archaea and bacteria in sediment of the pearl river estuary, china date: 2011-02-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3107-8 sha: doc_id: 12085 cord_uid: ubdzhkfq the diversity and abundance of ammonia-oxidizing archaea (aoa) and ammonia-oxidizing bacteria (aob) in the sediment of the pearl river estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qpcr). from one sediment sample s16, 36 aoa otus (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoa gene. among the 36 otus, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. for aob, only seven otus (based on 16s rrna gene) and eight otus (based on amoa gene) were obtained, showing lower diversity than aoa. the qpcr results revealed that aoa amoa gene copy numbers ranged from 9.6 × 10(6) to 5.1 × 10(7) copies per gram of sediment and aob amoa gene ranged from 9.5 × 10(4) to 6.2 × 10(5) copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the pearl river estuary were aoa. the terminal restriction fragment length polymorphism results showed that the relative abundance of aob species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the aob community composition. electronic supplementary material: the online version of this article (doi:10.1007/s00253-011-3107-8) contains supplementary material, which is available to authorized users. microorganisms in marine sediments contribute significantly to global cycles of organic and inorganic matters (whitman et al. 1998) , including nitrogen cycle. biological nitrogen fixation is the largest source of nitrogen to the ocean, whereas anoxic/anaerobic microbial processes are responsible for nitrogen losses (gruber and sarmiento 1997) . these two processes are connected by nitrification, the microbial oxidation of ammonia (nh 3 ) to nitrate (no 3 − ) via nitrite (no 2 − ). coupled nitrification/ denitrification can remove a substantial percentage (10~80%) of anthropogenic nitrogen pollution in estuaries (seitzinger 1988) . previous studies have focused on the ammonia-oxidizing bacteria (aob), mainly targeting both the 16s rrna gene and amoa gene which encodes α subunit of the ammonia monooxygenase (amo) (purkhold et al. 2000) . all the known aob are phylogenetically restricted to the βand γ-proteobacteria. recently, it was discovered that many archaea in the phylum crenarchaeota are also capable of performing ammonia oxidation like aob (könneke et al. 2005; venter et al. 2004 ). both ammonia oxidizing archaea (aoa) and aob contains the key enzyme amo which is composed of three subunits encoded by genes of amoa, amob, and amoc (klotz et al. 1997) and responsible for the conversion of ammonia to hydroxylamine. by performing the key first step in nitrification, both aoa and aob play an important role in the global nitrogen cycle. however, estuaries and their intertidal sediments experience large fluctuations in various hydrological and chemical conditions. therefore, the communities of aoa and aob might show dynamic changes in response to environmental factors. a few studies demonstrated that diverse aoa/aob phylotypes and distinct aoa/aob communities existed in different marine environments both on a large geographical scale and in local estuarine gradients (beman and francis 2006; francis et al. 2005; sahan and muyzer 2008) . studies already showed that archaeal amoa gene copy numbers were about 10 to 1,000 times of those of bacterial amoa in the north sea and north atlantic (wuchter et al. 2006) , indicating dominance of aoa in ocean environments. so far, most of the studies on aoa and aob in marine water and sediment focused on the coast of the east pacific ocean and atlantic. the diversity and spatial distribution of aoa and aob are still unknown in other ocean areas of the world, including the pearl river estuary. the pearl river, the third largest river in china, stretching for 2,214 km and draining an area of 452,000 km 2 , flows into the pearl river estuary through eight entrances. the pearl river estuary is located in the subtropical region in guangdong province, southern china. it represents one of the most important and complex ecosystems linking the highly developing land area and the south china sea. in recent years, the pearl river has a high load of anthropogenic nutrients from increased agricultural activities, fish dike farming, and wastewater runoff due to the increase in population and economic development in southern china and the pearl river delta region (huang et al. 2003) . previous study also showed high nitrification rates in summer (ammonia oxidation rate~1.5-33.1 μmol nl −1 day −1 , nitrite oxidation rate~0.6-32.0 μmol nl −1 day −1 ) (dai et al. 2008 ). however, the microorganisms responsible for the nitrification in this area are little known. in this study, five sediment samples were collected in the pearl river estuary from different sites along the salinity gradient (fig. 1) . the diversity of aoa and aob were revealed based on clone libraries. quantification of aoa and aob were performed using quantitative pcr (qpcr). for aoa, three primer sets of amoa gene were selected for construction of clone libraries and qpcr to evaluate the specificity and universality of these primers. terminal restriction fragment length polymorphism (t-rflp) was used to analyze the spatial distribution of different aob populations. surface sediment samples were collected from five locations along a transect from the pearl river estuary to the south china sea in the summer (june 2009; fig. 1 ) using a 250-cm 3 van veen grab (kc denmark, denmark). upon collection of the sediment samples, about 2 kg of sediment were transferred to sterile plastic bags and frozen at −80°c. frozen sediment samples were then transferred back to the laboratory with dry ice. except for the depth and temperature which were measured on site, other parameters were measured using the pore water of the sediments. concentrations of ammonium, nitrite, and nitrate were measured according to the standard methods (apha 2005) by nesslerization method, colorimetric method, and ultraviolet spectrophotometric screening method, respectively. for aob 16s rrna and amoa genes, pcr was performed in a total volume of 25 μl containing 20 ng of dna template, 2.5 μl buffer, 250 mm of each deoxyribonucleotide triphosphate, 1 u of extaq (takara) dna polymerase, and 0.2 μm of each primer. the major cycling program for each primer set was listed in table 1 . the presence and sizes of the pcr amplification products were determined by agarose (1%) gel electrophoresis. pcr products were purified by using quick-spin™ pcr products purification kit (intron biotechnology, seongnam-si, korea). then, the pcr products were cloned using inst/aclone™ pcr cloning kit (fermentas, vilnius, lithuania). white colonies were selected for insert screening by using pcr with primers m13f and m13r. the sequencing of selected clones was performed by using m13f primer on abi 3730xl capillary sequencers (pe applied biosystems, foster city, usa). the estimation of diversity index in each library was determined using the estimates v8.0 (http://viceroy.eeb. uconn.edu/estimates). the chao 1 estimator of species richness was calculated after 1,000 randomizations of sampling, without replacement. the percentage of coverage was calculated using good's method (good 1953) . operational taxonomic unit (otu) in this study was defined as a sequence group in which sequences differed by ≤3%. the nucleotide sequences were compared with those from the genbank using blastn in the national center for the biotechnology information (ncbi) server (http://www. ncbi.nlm.nih.gov). the sequences in this study and the reference sequences retrieved from the genbank were aligned by arb (http://www.arb-home.de) (ludwig et al. 2004 ) to construct phylogenetic trees using the neighborjoining method (based on jukes-cantor-corrected distance). bootstrap value was calculated based on 1,000 replications. quantitative pcr was performed using an icycler iq system (bio-rad, hercules, usa), three replicates for each sample. for aoa amoa gene, qpcr was conducted in a total volume of 30 μl containing 15 μl of failsafe™ pcr premix f, 5 μl of dna template, 0.2 μm of each primer, 1.5 u of amplitaq® dna polymerase, 15 mm mgcl 2 , and 0.5× sybr® green i (invitrogen, eugene, usa). for aob 16s rrna and amoa genes, pcr was performed in a total volume of 30 μl containing 15 μl of iq™ sybr® green voytek and ward 1995 cto654r ctagcyttgtagtt tcaaacgc kowalchuck et al. 1997 the concentration of dna was determined by nanodrop® spectrophotometer nd-1000 (thermo fisher scientific, usa). pcr was performed using failsafe™ pcr premix f (epicentre biotechnologies, madison, usa) following the cycling program for each primer set listed here a primers used for t-rflp analysis were labeled by hex super mix (bio-rad, hercules, usa), 5 μl of dna template, and 0.2 μm of each primer. the qpcr thermocycling steps were set as follows: 95°c for 4 min and 45 cycles at 95°c for 45 s, 55°c for 45 s, and 72°c for 45 s. cycling was completed by a final elongation step at 72°c for 10 min. the negative control containing no dna was subjected to the same procedure to exclude or detect any possible contamination. after qpcr assay, the specificity of amplification was verified by melting curve analysis and checking with agarose gel electrophoresis. for t-rflp, pcr was performed by using fluorescently labeled primer amoa-2r-hex for aob. pcr conditions were as the same as those described above for clone library construction. alui was selected to digest pcr products of aob amoa gene. the digestion mixture, containing 17 μl of purified pcr product (about 200 ng dna), 2 μl of buffer, and 1 μl of restriction enzyme (10 u/μl), was incubated at 37°c for 5 h. the fluorescently labeled t-rfs were run though an abi 3730xl capillary sequencers in the genescan mode. t-rflp data was analyzed using gene-marker v1.6 (softgenetics llc, pennsylvania, usa). because of the detection range of internal marker gs500, t-rfs smaller than 50 bp and larger than 500 bp were excluded from further analysis. the peaks were first selected by default parameters setting of the software genemarker with the threshold cutoff of peak intensity of 100. the relative abundance of each t-rf was then determined by calculating the ratio between the area of each peak and the total area of all peaks in one sample. the peaks with relative abundance less than 1% were neglected in this study. the sequences reported in this study were deposited in genbank under accession numbers gu988755-gu988900. physical and chemical properties of the sediment samples diversity of aoa amoa gene sequences archaeal amoa clone libraries were generated for the sediment sample s16 by using three primer sets (table 1) . a total of 145 clones were sequenced and grouped into 36 otus. in details, three clone libraries (a1, a2, and a3) recovered 19, 16, and 15 otus using primer sets i, ii, and iii, respectively (table s2 ). six otus (fig. 2a, otu1-6) were shared by the three clone libraries and accounted for 57%, 64%, and 70% of total clones in the three libraries, respectively. two otus (fig. 2a, otu7 and otu8) appeared in two clone libraries. the other 28 otus were only recovered in one of the three libraries. for aob 16s rrna gene clone library, as shown in fig. s1 , 26 clones were sequenced and grouped into seven otus, i.e., one otu in the genus nitrosospira and six otus in the genus nitrosomonas. similarly, 26 aob amoa gene sequences were generated from another clone library and grouped into eight otus (fig. 2b) : three otus in the genus nitrosospira and five otus in the genus nitrosomonas. no γ-aob sequences were found in the amoa gene clone library indicating that the major aob species in the samples were β-aob. to quantify the aoa abundance, three primer sets of aoa amoa gene were used for qpcr. the results showed that qpcr could be successfully conducted by using the primer sets i and iii. the qpcr results showed that the aoa abundance was significantly different by using two primer sets for the samples s9, s16, s19, and s21. generally, as shown in fig. 3 , the aoa abundance calculated from qpcr using primer set i was about 10 times higher than that using primer set iii. only for the sample s4, two primer sets resulted in the same level of aoa abundance. aoa amoa gene copy numbers based on qpcr by using primer set i ranged from 6.8×10 3 to 2.2×10 4 copies per nanogram dna and from 9.6×10 6 to 5.1×10 7 copies per gram of sediment (wet weight). over all, there was no significantly difference among five sediment samples considering the variation of qpcr itself. aob abundance was quantified by qpcr using both aob 16s rrna and amoa genes. as shown in fig. 3 , aob 16s rrna gene ranged from 15 to 126 copies per nanogram dna and 2.7×10 4 to 1.4×10 5 copies per gram of sediment. similarly, aob amoa gene resulted in 67 to 333 copies per nanogram dna and 9.5×10 4 to 6.2×10 5 copies per gram of sediment. if assuming that there is one copy of 16s rrna gene per cell for both nitrospira and nitrosomonas (dionisi et al. 2002) , the mixed aob community in this study should contain one copy 16s rrna gene per cell, according to the clone library results. assuming that there are 2.5 copies of amoa gene per aob cell (norton et al. 2002) , thus the copy number of aob amoa gene in one sample should be 2.5 times that of aob 16s rrna gene. for five sites, the average copy numbers per gram of sediment were 2.9×10 5 and 1.2×10 5 for aob and 16s rrna gene, respectively. the results of cells of fig. 2 phylogenetic trees of aoa (a) and aob (b). the neighbor-joining trees were constructed using arb based on jukes-cantor-corrected dna distances, showing the phylogenetic affiliation of amoa gene sequences from the pearl river estuary sediment s16 (printed in bold) and from other environmental samples or pure strain retrieved from the genebank. in (a), except for the singletons, one clone was selected from each otu for phylogenetic analysis and the total number of clones grouped to that otu was listed after the clone name. the bootstraps (based on 1,000 replications) larger than 60% were indicated by the black dots aob based on amoa gene copy number were generally consistent with those based on 16s rrna gene (fig. 3) . t-rflp analysis clearly showed the dynamic changes of aob community among five sampling sites. there were total of four t-rfs appearing in profiles. based on the relative abundance of each one, t-rfs of 95 and 264 bp represented the dominant aob and other two t-rfs of 143 and 486 bp only occupied very low abundance, less than 6% individually. all the four t-rfs could be correlated to the clones of aob amoa gene clone library, as shown in table s3 . the t-rf of 95 bp corresponded to aob in the genus nitrosomonas, while t-rf of 264 bp was mainly derived from aob in the genus nitrosospira, although several nitrosomonas clones also resulted in this t-rf. the clone library was constructed using the sediment sample s16, and here, it was assumed that the same t-rfs in other four samples represented similar aob species as in the sample s16. obviously, as shown in fig. 4 , the species compositions of aob among five samples were dramatically different. gradient changes showed along the sample collection sites. from the sites s4 to s21, the relative abundance of nitrosomonas, indicated by t-rf of 95 bp, gradually decreased from 58% to the level under detection by t-rflp. in contrast, the relative abundance of aob in the genus nitrosospira, indicated by t-rf of 264 bp, significantly increased to double. it was also possible that the same t-rf in different samples came from different aob species, or the t-rf of 264 bp in other samples was composed by aob in two genera in different ratios, thus, in fact the diversity changes of aob community among the sampling sites should be even more complicated than that observed here. phylogenetic analysis indicated that most of aoa amoa gene sequences in this study were closely related to uncultured sediment aoa found at other places, except fig. 2 (continued) three small clusters (clusters i, ii, and iii). clusters i and iii were grouped with sequences from mixed environmental samples, including soil (ab529723, gq481040), hot spring (gq226087, gq226097), sediment (fj951725, eu885576), active sludge of bioreactor (eu860263), and the identified pure aoa species nitrosopumilus maritimus (eu239959). in cluster ii, the two sequences were affiliated to those obtained from freshwater samples. phylogenetic tree also showed that the most closely related sequences of five shared otus, except otu6, were all from the san francisco bay estuary sediments (mosier and francis 2008) . in total, these five otus reached 57.2% abundance (83 out of 145 clones) in the sediment of this study. these results indicated that the aoa represented by otu1 to otu5 in this study, and their relatives from other estuaries were possibly major aoa population in the estuary sediments, although so far, these aoa are still quite difficult to be cultured and identified. among them, otu1 represented the most dominant aoa group in this study, occupying 25.5% relative abundance (37 of 145 clones). for aob, generally, the results from two clone libraries were comparable and consistent. it was also consistent with previous reports that aob in two genera nitrosomonas and nitrosospira were the dominant aob in estuarine sediments (mosier and francis 2008) . compared to aoa amoa gene, aob showed lower diversity for the same sample in this study. the clone library and agarose gel electrophoresis results indicated that both primer sets did not generated unspecific pcr products; thus, it seems that the real-time pcr results from primer set i should be more accurate than that from primer set iii. the difference might be due to the different binding efficiencies of the two forward primers to dna templates as the reverse primers are the same. so far, quantification of amoa gene abundance using qpcr is still in a relatively early stage of development, and more reliable measurement methods must be applied to confirm the previous findings. compared with the results of mosier and francis (2008) , the aoa abundance in this study was at the high end of that in san francisco bay estuary sediments (from 1.4×10 4 to 3.9×10 7 copies per gram of sediment). however, in their study, high aoa abundance only occurred in low-salinity regions (0.2-9 psu). the salinities of five sampling sites in this study increased from 5.6 to 24.6 psu, while the aoa abundance kept at high level along the salinity gradient. it might due to the high ammonia concentrations at these sites. these results also provide the possible reason for the previous report of high nitrification rates in summer at the pearl river estuary (dai et al. 2008) . it was reported that aob abundance increased with salinity (mosier and francis 2008) . however, in this study, the results did not show the same relationship. overall, aob amoa gene copy number was 100 times lower than that of aoa, suggesting that the dominant ammonia-oxidizing microorganisms in the sediment of the pearl river estuary were aoa. comparing the environmental parameters of five sites, the salinity ranged from 5.6 to 24.6 psu, which possibly was the key factor shaping the aob community composition, although total abundance of aob did not show significant fluctuation. the results indicated that aob in the genus fig. 4 relative abundance of aob t-rfs in the pearl river estuary sediments, determined by the corresponding normalized t-rflp peak area. pcr was performed by using fluorescently labeled primer amoa-2r-hex for aob. pcr products were digested using alui. the relative abundance of the selected t-rf peak (intensity above 100) was determined by calculating the ratio of its area and the total area of all selected peaks in one sample nitrosomonas in this study might prefer low salinity, while in high salinity, the species in nitrosospira were the dominant aob. it was consistent with the previous findings in the chesapeake bay (francis et al. 2003) , plum island sound , ythan (scotland) estuaries (freitag et al. 2006) , and the san francisco bay estuary (mosier and francis 2008) , where similar distribution patterns of aob in two genera were reported. t-rflp analysis was also applied to investigate the dynamic shift of aoa community. however, it was found that there were only three major peaks in the profiles (fig. s3 ). the number of t-rfs was largely smaller than that of otus in aoa clone library. it means the resolution of the method was too low. the sequences of aoa amoa gene obtained in this study were aligned and virtually digested by over twenty restricted enzymes. but, there was not any enzyme which separated these sequences very well. it was concluded that t-rflp was not a suitable method to analyze the difference of aoa community in this study. selection of primers for aoa amoa gene junier et al. (2010) summarized all the published primer sets for aoa amoa gene amplification. the primer selection is a key step for microbial community study based on pcr. however, the specificity and sensitivity of these primers were not compared so far. in this study, three primer sets for aoa amoa gene were selected for comparison. the results of clone libraries showed that both shared and distinct clones were recovered by using different primer sets. generally, by using any one of three primer sets, the dominant aoa species in this sediment sample, represented by the shared six otus, could be recovered from the clone library. however, those distinct otus might be recovered only by specific primers. specially, the sequences in cluster i were only amplified by primer sets ii and iii. of course, the difference among three clone libraries was also possibly due to the low abundance of those otus and insufficient coverage of the clone library. comparison of chao 1 among three clone libraries showed that the primer set iii resulted in the highest value and significantly higher than the primer set ii, indicating that the primer set iii might match more aoa amoa gene sequences than other two primer sets. theoretically, using this primer set iii may recover the highest diversity of aoa amoa genes in this sediment sample, although we got the highest number of otu from clone library by using primer set i. the significant difference was also shown when primer sets were applied in qpcr. our previous aoa study in activated sludge already reported that two primer sets showed impact in clone library results (zhang et al. 2009 ). these results suggested that two or more primer sets should be tried when starting the pcr for a new environment sample. it was also indicated that the primer selection was critical for clone library construction, qpcr, and other methods based on pcr. diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: bahiadel tobari loss of diversity of ammonia-oxidizing bacteria correlates within creasing salinity in an estuary system nitrification and inorganic nitrogen distribution in a large perturbed river/estuarine system: the pearl river estuary cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol quantification of nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrospira spp. from full-scale wastewater treatment plants by competitive pcr diversity of ammonia monooxygenase (amoa) genes across environmental gradients in chesapeake bay sediments ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient the population frequencies of species and the estimation of population parameters global patterns of marine nitrogen fixation and denitrification pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine crenarchaeota the characteristics of nutrients and eutrophication in the pearl river estuary, south china phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (aom) in the environment a gene encoding a membrane protein exists upstream of the amoa/amob genes in ammonia-oxidizing bacteria; a third member of the amo operon isolation of an autotrophic ammonia-oxidizing marine archaeon analysis of ammonia-oxidizing bacteria of the β subdivision of the class proteobacteria in costal sand dunes by denaturing gradient gel electrophoresis and sequencing of pcr-amplified 16 s ribosomal dna fragments arb: a software environment for sequence data relative abundance and diversity of ammonia-oxidizing archaea and bacteria in san francisco bay estuary diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria phylogeny of all recognized species of ammonia oxidizers based on comparative 16 s rrna and amoa sequence analysis: implications for molecular diversity surveys the ammonia monooxygenase structural gene amoa as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations diversity and spatio-temporal distribution of ammonia-oxidizing archaea and bacteria in sediments of the westerschelde estuary denitrification in fresh water and coastal marine ecosystems: ecological and geochemical significance environmental genome shotgun sequencing of the sargasso sea detection of ammonium-oxidizing bacteria of the beta-subclass of the class proteobacteria in aquatic samples with the pcr prokaryotes: the unseen majority archaeal nitrification in the ocean occurrence of ammonia-oxidizing archaea (aoa) inactivated sludges of a laboratory scale reactor and two wastewater treatment plants the authors wish to thank the hong kong general research fund (hku7197/08e) for the financial support of this study, and lin ye wish to thank hku for the postgraduate studentship. this work was partially supported by the cas/safea international partnership program for creative research teams to py qian.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012101-mfgca1ou authors: luesken, francisca a.; van alen, theo a.; van der biezen, erwin; frijters, carla; toonen, ger; kampman, christel; hendrickx, tim l. g.; zeeman, grietje; temmink, hardy; strous, marc; op den camp, huub j. m.; jetten, mike s. m. title: diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge date: 2011-06-11 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3361-9 sha: doc_id: 12101 cord_uid: mfgca1ou recently discovered microorganisms affiliated to the bacterial phylum nc10, named “candidatus methylomirabilis oxyfera”, perform nitrite-dependent anaerobic methane oxidation. these microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. to find suitable inocula for reactor startup, ten selected wastewater treatment plants (wwtps) located in the netherlands were screened for the endogenous presence of m. oxyfera using molecular diagnostic methods. we could identify nc10 bacteria with 98% similarity to m. oxyfera in nine out of ten wwtps tested. sludge from one selected wwtp was used to start a new enrichment culture of nc10 bacteria. this enrichment was monitored using specific pmoa primers and m. oxyfera cells were visualized with fluorescence oligonucleotide probes. after 112 days, the enrichment consumed up to 0.4 mm no(2)(−) per day. the results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature. electronic supplementary material: the online version of this article (doi:10.1007/s00253-011-3361-9) contains supplementary material, which is available to authorized users. anaerobic nitrite-dependent methane oxidation is a recently discovered process performed by bacteria with doubling times of approximately 1-2 weeks (raghoebarsing et al. 2006; ettwig et al. 2008) . the dominant bacteria present in the anaerobic enrichment cultures were members of the nc10 phylum (ettwig et al. 2009; hu et al. 2009 ). the genome of this dominant bacterium, named "candidatus methylomirabilis oxyfera", could be assembled from metagenomic data resulting in a 2.7-mb circular single chromosome which contained genes of both anaerobic and, electronic supplementary material the online version of this article (doi:10.1007/s00253-011-3361-9) contains supplementary material, which is available to authorized users. surprisingly, aerobic metabolic pathways (ettwig et al. 2010) . the genome harbored the complete aerobic pathway to oxidize methane, including the pmocab operon encoding the particulate methane monooxygenase (pmmo) complex for which recently pcr primers were developed (luesken et al. 2011) . conversely, the denitrification pathway was not complete. genes nosdfyz encoding nitrous oxide reductase were missing from the genome and nosl appeared not to be expressed, indicating that another denitrifying pathway had to be operative (wu et al. 2011) . dedicated stable isotope studies showed that this organism could make its own molecular oxygen from nitrite via nitric oxide (ettwig et al. 2010) . the produced oxygen was mainly used to oxidize methane in an anaerobic environment according to the expected stoichiometry: 3 ch 4 þ 8 no 2 à þ 8 h þ ! 3 co 2 þ 4 n 2 þ 10 h 2 o (eq. 1) (wu et al. 2011) . anaerobic wastewater treatment compared to conventional aerobic processes has advantages like a reduced production of sludge, a smaller footprint, and the production of biogas (methane) which can be used as an energy source (van haandel and lettinga 1994; lema and omil 2001; aiyuk et al. 2006) . one of the most established anaerobic techniques is the upflow anaerobic sludge blanket (uasb) first described by lettinga et al. (1980) . in the absence of oxygen, the microbial community, present in the uasb reactor, degrades organic matter eventually into the main products ammonium and methane (toerien and hattingh 1969) . the produced ammonium should be removed in accordance with the stringent rules for nitrogen compounds in wastewater effluent (http://ec.europa.eu/ environment/water/water-urbanwaste/index_en.html). methane contributes to the greenhouse effect when released to the environment and should therefore be removed or used in an energy-efficient way (cakir and stenstrom 2005; bogner et al. 2008) . m. oxyfera-type bacteria could use methane to drive denitrification, circumventing the purchase of electron donors like methanol for nitrogen removal. to obtain sufficient oxidized nitrogen for methane oxidation by m. oxyfera-type bacteria, partial nitrification of ammonium could be used (van dongen et al. 2001 ). this makes m. oxyfera-type bacteria important candidates for a novel way of sustainable anaerobic wastewater treatment. to date, enrichment cultures of m. oxyfera have been obtained from two different freshwater systems in the netherlands (raghoebarsing et al. 2006; ettwig et al. 2009 ). in addition, enrichments of nc10 bacteria were obtained from a mixed sample of freshwater sediment, anaerobic sludge, and return activated sludge (hu et al. 2009 ). in the present study, we detected m. oxyfera-type bacteria in nine out of ten screened wastewater treatment plants (wwtps) located in the netherlands using 16s rrna gene analysis. one of these wwtps was selected and biomass was used to start an enrichment culture of m. oxyfera. at the end of the experimental period, this enrichment was capable of nitrite-dependent methane oxidation with conversion rates of 0.3 nmol ch 4 min −1 mg protein −1 and 0.9 nmol no 2 − min −1 mg protein −1 at ambient temperature. all the wwtp sludge samples used for screening came from plants located in the netherlands (table 1 ). the wwtps were selected based on a sludge retention time ≥8 days and bod/n ≤4.7. the wwtp in lieshout treats industrial water from all samples we obtained 16s rrna clone libraries (clone libraries are named after the sampling location), except the wwtp in varsseveld a biomass from this wwtp was used to inoculate a bioreactor. four samples originating from the lieshout treatment plant were analyzed using molecular methods: inoc lieshout, enr lieshout, enr 332 day lieshout, and wwtp lieshout using uasb reactors followed by a carrousel. the other nine wwtps in our selection process wastewater originating from both domestic and industrial sources. these treatment plants make use of pre-denitrification in combination with an activated sludge system. besides this system, reject water of a digester is processed by the wwtp in kralingseveer and the wwtp in lichtenvoorde receives effluent of a process where anaerobically treated wastewater of a tannery is combined with partial nitrification and anaerobic ammonium oxidation (anammox). the wwtps haarlo, varsseveld, and heerenveen apply chemical phosphorus removal. samples from the activated sludge were taken for molecular analysis. the industrial water treatment plant in lieshout (bavaria b.v., lieshout, the netherlands) uses three separate uasb reactors. subsequently, the uasb effluent is treated by a carrousel with two aeration points. the sludge samples for molecular screening and inoculation of a bioreactor were taken at the end of the carrousel after the second aeration point. sludge (2 l) was mixed with ambient water to inoculate the bioreactor. dna from the lieshout wwtp and the enrichment was extracted and purified according to ettwig et al. (2009) . dna from the other nine wwtp samples was extracted using the powersoil® dna isolation kit (mo bio, carlsbad, ca, usa). finally, dna was dissolved in diethylpyrocarbonate (depc) treated water (invitrogen uk) and the quality was checked with gel agarose electrophoresis. to analyze biodiversity of nc10 phylum bacteria, the isolated dna was used as a template for a 16s rrna targeted pcr. the 16s rrna primers used in the nested pcr approach were nc10 specific forward primer 202f (ettwig et al. 2009 ) and general bacterial reverse primer 1545r (juretschko et al. 1998 ). thermal cycling was performed with an initial melting step for 4 min at 94°c, followed by 35 cycles of denaturation at 94°c for 1 min, annealing at 57°c for 1 min, and elongation at 72°c for 2.5 min. finally, an elongation step at 72°c for 10 min was performed. the pcr product obtained was used as template for a nested pcr using the nc10 specific primers qp1f and qp2r, originally developed for qpcr (ettwig et al. 2009 ). in the nested pcr, the thermal cycling was performed as described above, with an annealing temperature of 65°c for 1 min. for the clone library of the 332day lieshout enrichment, we used an annealing temperature gradient (50-60°c). to identify the m. oxyfera-type bacteria on a functional level, four different primer combinations targeting the pmoa gene were used. four different dna samples originating from the inoculum (inoc) and enrichment (enr) of the lieshout treatment plant (inoc lieshout, enr lieshout, enr 332 day lieshout, and wwtp lieshout) served as a template for pcr and subsequently pmoa gene libraries were constructed. first, novel forward primer a189_b (luesken et al. 2011 ) with general reverse primer 682r (holmes et al. 1995) were used in a direct pcr. secondly, the forward primer a189_b and reverse primer cmo682 (luesken et al. 2011) were used in a nested pcr approach with m. oxyfera specific primers cmo182 and cmo568 (luesken et al. 2011) . the third primer combination was forward primer a189_b combined with reverse primer cmo682, and in the fourth combination primers cmo182 and cmo568 were used. the third and fourth primer combinations were used in a direct pcr on dna samples of the enrichment obtained from lieshout sludge. for all the four primer combinations used, thermal cycling was performed according to luesken et al. (2011) . all pcr reactions were performed with the quanta bioscience inc., perfecta® sybr® green fastmix® 76 (gaithersburg, md, usa). the pcr products were cloned with the pgem®-t easy cloning kit (promega usa) and xl1 blue escherichia coli competent cells. a maximum of about 20 clones per library were selected. subsequently, the plasmids were isolated using the genejet miniprep kit (fermentas, lithuania). the diagnostics center of nijmegen university medical center performed sequencing using m13 forward and reverse primers. the quality of the sequences was checked using the chromas lite (version 2.01) program. to obtain related sequences (>93% similarity) from genbank (http://www.ncbi.nlm.nih.gov/genbank/), a blast search was performed. representative 16s rrna and pmoa sequences from each location were submitted to genbank (accession numbers jf706170-jf706214). the sequences were aligned using mega4 software (tamura et al. 2007) , and manually checked and trimmed. phylogenetic trees were calculated in mega with the neighbor-joining method and with the pairwise deletion option for gaps. the tree topology was tested by bootstrap analysis (1,000 replicates). bioreactors of 15 l and 3 l (applikon, schiedam, the netherlands) were used for cultivation. for efficient biomass retention, the bioreactors were operated in a sequencing-batch mode (strous et al. 1998 ). the settling cycle consisted of 23.2 h of constant medium supply, 20 min of settling, and 30 min of pumping out the excess liquid. poorly settling biomass was collected using an external settler (2.3 l). during medium supply (flow rate 0.3-1 lday −1 ), the reactor was stirred at 100 rpm and sparged with ch 4 -co 2 (95:5 vol/vol; purity >99.995%; flow rate 10 ml min −1 ; air liquide, france). the bioreactors were equipped with a level controller. the medium, in both reactor systems, was continuously sparged with ar-co 2 (95:5, vol/vol) to maintain anoxic conditions and contained the following components (per liter): khco 3 , 0.5 to 1 g; kh 2 po 4 , 0.05 g; cacl 2 ·2h 2 o, 0.3 g; mgso 4 ·7h 2 o, 0.2 g; nano 3 , 0.035 to 0.213 g (0.5 to 3 mm); nano 2 , 0.0069 to 0.41 g (0.1 to 6 mm); an acidic trace element solution, 0.5 ml; and an alkaline trace element solution, 0.2 ml. the acidic (100 mm hcl) trace element solution contained (per liter) 2.085 g feso 4 ·7h 2 o, 0.068 g znso 4 ·7h 2 o, 0.12 g cocl 2 ·6h 2 o, 0.5 g mncl 2 ·4h 2 o, 0.32 g cuso 4 , 0.095 g nicl 2 ·6h 2 o, and 0.014 g h 3 bo 3 . the alkaline (10 mm naoh) trace element solution contained (per liter) 0.067 g seo 2 , 0.050 g na 2 wo 4 ·2h 2 o, and 0.242 g na 2 moo 4 . all medium components were sterilized, either by 0.2 μm filtration (acidic trace element solution) or by autoclaving. both bioreactors (15 l and 3 l) were equipped with a clark-type oxygen electrode and ph electrode (continuously monitored) and kept at ambient temperature (20-23°c). the ph of the cultures liquid varied between 6.8 and 7.3. the bioreactors were wrapped in black foil and black viton and norprene tubing with low oxygen permeability was used (cole parmer, usa). when the nitrite concentration was <0.1 or >1 mm, the medium flow was adjusted (influent varied between 0.3 and 1 lday −1 ). in addition, nano 2 and khco 3 concentrations in the medium varied depending on the denitrifying activity of the culture. both bioreactors were operated aseptically. the external settler (2.3 l schott bottle), coupled to the 15l bioreactor, was removed from the effluent tubing and operated as batch incubation. to obtain a headspace of 0.3 l, ar/co 2 was pumped into the settler and the excess fluid was discarded. two liters of liquid culture remained in the settler and ch 4 was added until a final concentration in the headspace of 4%. on t=0 h, 10 mg l −1 no 2 − and 100 mg l −1 no 3 − were present in the settler. samples were taken on a regular basis until 68 h. to determine the activity in the 3-l bioreactor, batch incubations were performed at t=308 and 315 days. the methane flow was stopped and the headspace (400 ml) of the bioreactor was flushed with ar/co 2 (10 ml min −1 ) for 30 min yielding a ch 4 concentration in the headspace of 5.3% and 4.8%, respectively. for the incubation at t= 308 days, the medium flow was stopped and nitrite was added to a final concentration of 1.3 mm in the bioreactor. measurements were started after a stabilization period (overnight). at t=315 days, a second activity test was performed with an initial nitrite concentration of 0.2 mm. in both incubations, methane, nitrite, and nitrate samples were taken (until t=10 h) and analyzed as described below. biomass samples (2 ml) were taken from the enrichment culture and centrifuged. the pellet was washed in phosphate-buffered saline (pbs; 10 mm na 2 hpo 4 / nah 2 po 4 ph 7.5 and 130 mm nacl) and fixed for 2 h on ice in 3% (w/v) paraformaldehyde in pbs. after incubation, the samples were washed with pbs and resuspended in ethanol and pbs (1:1). the fixed sample was stored at −18°c. fixed biomass (10 μl) was spotted on teflon-coated microscopic slides and dehydrated for 3 min in subsequently 50%, 80%, and 96% ethanol. the probes were hybridized for 1.5 h at 46°c in hybridization buffer (900 mm nacl, 2 mm tris/hcl ph 8.0, 0.2‰ sodium dodecyl sulfate) and 50% formamide. the following oligonucleotide probes were used: s-*-dbact-0193-a-a-18 (dbact193) and s-*-dbact-1027-a-a-18 (dbact1027), specific for bacteria affiliated with the nc10 phylum (raghoebarsing et al. 2006) ; and s-d-bact-0338-a-a-18 (eub338), specific for most bacteria (amann et al. 1990 ). the slides were examined using a zeiss axioplan ii epifluorescence microscope with digital video camera and image analysis software (axiovision, zeiss, germany). for routine nitrite analysis, merckoquant test strips (0 to 80 mg l −1 nitrite; merck, germany) were used. nitrite and nitrate samples (1 ml) from the batch experiments were measured colorimetrically (kartal et al. 2006 ). the total protein content was determined by the bca (bicinchoninic acid assay; pierce, usa) according to the manufacturer's protocol. bovine serum albumin (thermo scientific, usa) was used as a standard. the methane concentration was determined by gas chromatography (ettwig et al. 2008) . to find suitable inocula to start pilot scale tests for application of nitrite-dependent anaerobic methane oxidation, ten wwtps in the netherlands were screened with molecular tools (table 1 ). all wwtps treat wastewater originating from both domestic and industrial sources, except the wwtp in lieshout. this plant treats only industrial (brewery) water at moderate temperatures. relatively long sludge retention times and low bod/n ratios were used as criteria to select the wwtps for screening. these criteria in combination with alternating oxicanoxic conditions present in all wwtps might reflect the niche of m. oxyfera bacteria where both methane and oxidized nitrogen compounds are present at the same time. a molecular survey was conducted to screen for m. oxyfera-type bacteria after dna extraction from the sludge samples of these wwtps. to detect nc10 bacteria, a direct pcr with nc10 specific primer 202f and general primer 1545r targeting the 16s rrna gene was performed on one of the wwtp sludge samples (lieshout). this resulted in a pcr product of the right size, but this product was hardly visible with gel electrophoresis (data not shown). therefore, a nested pcr approach was performed on all wwtp sludge samples. in nine out of ten wwtps tested, m. oxyfera-type bacteria could be detected (fig. 1a) . m. oxyfera-type bacteria were not detected in the wwtp in varsseveld. recently, pcr primer sets for detection of m. oxyfera pmoa genes were published (luesken et al. 2011) . these sets were used in the later stage of this study (see below) complementary to the 16s rrna approach. soil c horizon baccs (eu335172, eu335192); uranium mill tailing colorado en new mexico (aj519670, aj519650); ab486305 rice paddy soil japan; ab426228 benzene-degrading enrichment japan; fj748807 pearl river estuary china; ef92972 iron-manganese nodule soil 0.02 a fig. 1 a phylogenetic tree of 16s rrna gene sequences of the nc10 phylum, including sequences of nine different wwtps located in the netherlands (bold). the phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. all sequences found are represented in group a of the nc10 phylum (group a, b, c, and d are described in ettwig et al. 2009 ). b phylogenetic tree of pmoa sequences including clones from inoc lieshout, enr lieshout, enr 332 day lieshout, and wwtp lieshout. the phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. the clones indicated with "a" were obtained with the primers a189_b and 682r, clones indicated with "b" were obtained with a nested approach using primers a189_b and cmo682 as a template for primers cmo182 and cmo568. clones obtained with a direct pcr using the primers a189_b and cmo682 are indicated with "c". clones obtained with primers cmo182 and cmo568 are indicated with "d" the industrial wastewater treatment plant in lieshout was selected to start an enrichment culture. important treatment processes of this plant are three uasb reactors in parallel followed by a carrousel for nitrification and denitrification. in the uasb reactor, organic compounds are degraded and the produced methane is collected and used as an energy source by the brewery, but dissolved methane is difficult to recover and remains in the liquid phase. the effluent from the uasb reactors contains reduced nitrogen compounds (0.046±0.014 gl −1 n, of which 0.007±0.007 gl −1 is nh 4 + ). this effluent is treated in the carrousel that makes use of two aeration points, resulting in oxic and anoxic zones where nitrification and denitrification can take place (hydraulic retention time is 4 days). compared to the other screened wwtps, the carrousel in this plant has a prolonged sludge retention time (50 days). the presence of both dissolved methane and oxidized nitrogen compounds (nitrate and nitrite), and the prolonged sludge retention time might favor m. oxyfera bacteria. samples to start an enrichment culture were taken after the second aeration point in the carrousel. a 15-l sequencing batch reactor was inoculated with biomass from the wwtp lieshout. during the initial trial period, we noticed that the denitrifying activity measured ub schoehsee sediment germany (ef623812); uncultured methanotroph tundra soil (af547175, af547181, af547178) crenothrix polyspora 3 clones (dq295901, dq295902, dq295904) beta proteobactria ammonium oxidizers (ef175100, u76553, dq228465, al954747, af037107, af042171) gamma proteobacteria pxma (eu722433, eu722432, eu722430, eu722431) fig. 1 (continued) inside the bioreactor gradually decreased. furthermore, biomass that was not efficiently retained in the reactor accumulated in the external settler. the biomass collected in the external settler (0.7 l liquid culture containing 1.5 g of protein) did show denitrifying activity: no 2 − and no 3 − were converted at rates of 2.1 and 9.2 nmol h −1 mg protein −1 , respectively. besides the denitrification, anaerobic methane oxidizing activity (1.7 nmol ch 4 h −1 mg protein −1 ) could be measured. in addition to these activity experiments, the biomass in the external settler was analyzed for the presence of nc10 bacteria using a nested pcr approach. m. oxyfera-type bacteria were detected in the settler biomass and were all clustering in group a of the nc10 phylum (ettwig et al. 2009 ). the sequences obtained from the external settler were closely related to the 14 sequences found in the inoculum originating from the wwtp in lieshout (fig. 1a) . based on these results, the biomass from the external settler was used to inoculate a new 3-l bioreactor (sbr) that was operated at ambient temperature under strict anoxic conditions and a stringent biomass retention regime. at the startup of the 3l bioreactor, the initial nitrite concentration (1 mm) was diluted to 0.01 mm by pumping fresh medium into the reactor (day 0-50). the activity of the culture till 77 days was low but thereafter the culture showed increasing denitrifying activity up to 0.4 mm of no 2 − day −1 on t= 112 days (fig. 2a ). there were a number of technical problems (power failures, interruption of the methane supply) from days 112 to 175 causing fluctuating nitrite and methane consumption activities. however, from day 175, there was an ongoing increasing nitrite reducing activity up to 1.1 mm day −1 (fig. 2a) . nitrite-dependent anaerobic methane oxidation activity measurements were performed with the enrichment culture on t=308 and 315 days. the anaerobic conversion of nitrite and methane was measured in the 3-l bioreactor. at that time, the protein concentration in the enrichment culture was 0.4 gl −1 . nitrite was added to the enrichment culture prior to the experiment with a final concentration of 1.3 mm. the measured anaerobic methane oxidizing activity was 0.3 nmol ch 4 min −1 mg protein −1 . nitrite was converted with 0.9 nmol no 2 − min −1 mg protein −1 (fig. 2b) . in this study, the measured stoichiometry for methane to nitrite was 3 ch 4 :9.8 no 2 − , this value was close to theoretical value 3 ch 4 :8 no 2 − (raghoebarsing et al. 2006; ettwig et al. 2009 ). after 315 days, the conversion rates for methane and nitrite were 0.5 nmol min − 1 mg protein − 1 and 1.2 nmol min −1 mg protein −1 , respectively (data not shown). the measured stoichiometry was 3 ch 4 :10.1 no 2 − . to investigate the biodiversity in the enrichment after 332 days, the culture was analyzed using specific nc10 primers targeting the 16s rrna gene. the sequences obtained from this enriched biomass were highly similar to the 16s rrna gene sequences found in the inoculum and were all clustering in group a of the nc10 phylum (ettwig et al. 2009 ). during cultivation, the nc10 members of group a were predominantly enriched (fig. 1a) . similar observations were made in a previous study (ettwig et al. 2009 ) and suggested that the nc10 bacteria present in group a performed nitrite-dependent anaerobic methane oxidation. in addition to the phylogenetic analysis after 332 days, samples directly taken from the lieshout wwtp (1 year after taking the inoculum) were screened. sequences retrieved from the wwtp in lieshout (six clones), 1 year after taking the inoculum, were all clustering in group b of the nc10 phylum (ettwig et al. 2009 ). there were no sequences found in group a of the nc10 phylum, unlike the results of the original inoculum where 14 sequences clustered in group a (fig. 1a) . since the genome of m. oxyfera contained the complete pathway to oxidize methane aerobically, pmoa could serve as a functional marker. the pmoa gene encodes one of the subunits of the pmmo complex that facilitates the aerobic conversion of methane to methanol. specific pmoa primers were recently developed to monitor and identify nc10 bacteria on a functional level (luesken et al. 2011) . the four different dna samples (originating from the lieshout treatment plant) analyzed for 16s rrna were also used as a template testing four different primer combinations targeting the pmoa gene. first, a newly developed primer a189_b (luesken et al. 2011) was combined with the commonly used 682r primer (holmes et al. 1995) . all pmoa sequences retrieved in this study using this primer combination (clone libraries indicated with "a"), clustered with either methylococcus capsulatus (gammaproteobacteria) or crenothrix polyspora (gammaproteobacteria) and not with m. oxyfera (fig. 1b) . secondly, the primer combination a189_b and cmo682 was used in a nested pcr approach with novel primers cmo182 and cmo568 as described by luesken et al. (2011) to detect pmoa sequences related to m. oxyfera. all four dna samples were analyzed using this combination (clone libraries indicated with "b") and contained sequences clustering with pmoa gene sequences of m. oxyfera (fig. 1b) . in addition to these two experiments, clone libraries for the detection of m. oxyfera in the enrichment were made using a direct specific pcr with either primer combination a189_b and cmo682 (clone libraries indicated with "c") or cmo182 and cmo568 (indicated with "d"). although the third combination with primers a189_b and cmo682 resulted in a pcr product with settler dna as the template, only one pmoa sequence (out of 13) was obtained after cloning and sequencing. the dna extracted after 332 days of enrichment (fig. 1b) yielded 12 pmoa sequences (out of 12 clones screened). the fourth and last combination was a direct pcr with pmoa primers cmo182 and cmo568 using dna from the 332-day enrichment as template. all clones obtained were similar to the pmoa gene sequence of m. oxyfera (fig. 1b) . in order to get an impression of the relative abundance of m. oxyfera cells compared to the other community members, fluorescence in situ hybridization (fish) was performed using general and specific probes (fig. 3) . no m. oxyfera-type bacteria could be detected in the inoculum (fig. 3a) , probably because the number of cells was below detection level (amann et al. 1995) . this is consistent with the 16s rrna and pmoa phylogenetic analysis (see above). although pcr is a more sensitive technique compared to fish, a nested pcr approach was necessary to detect m. oxyfera-type bacteria in the inoculum. after 64 days of a b c fig. 3 increasing population of m. oxyfera-type bacteria in lieshout enrichment culture, visualized with fish. the cells were hybridized with the probes s-*-dbact-1027-a-a-18 specific for the nc10 phylum (red) and s-d-bact-0338-a-a-18 that target most, but not all bacteria (dark blue). a there were no m. oxyfera cells detected in the inoculum, using fish. b after an incubation period of 64 days in a 3-l reactor, m. oxyfera-type bacteria were enriched for~2-3% (represented in pink, caused by double hybridization of dbact1027 and eub338). c clusters and some detached cells of m. oxyfera-type bacteria are present in the culture after 308 days (represented in pink). m. oxyfera-type bacteria were enriched for~60-70%. probe s-*-dbact-0193-a-a-18 specific for m. oxyfera bacteria showed the same result. scale bar is 20 μm cultivation in the 3-l bioreactor, approximately 2-3% of the microbial community hybridized with specific probes for m. oxyfera (fig. 3b) . after 308 days, the m. oxyfera population was enriched for approximately 60-70% (fig. 3c) . the enriched bacteria appeared to grow mainly in clusters, but some single cells were also visible as observed previously (ettwig et al. 2009 ). ten wwtps were screened with molecular tools to find suitable inocula to start pilot scale tests for application of nitrite-dependent anaerobic methane oxidation. in this study, nc10 specific primers targeting the 16s rrna gene were used to identify m. oxyfera-type bacteria in the selected wwtps. it appeared to be that a nested pcr approach was necessary to detect nc10 bacteria in the wwtps sludge samples. apparently, it was difficult to amplify the 16s rrna gene from nc10 bacteria using a direct pcr, probably caused by low amounts of dna from nc10 bacteria in the tested sludges. this is in contrast with previous studies using freshwater sediment samples, in which a direct pcr with 202f and 1492r resulted in strong pcr bands of the right size and subsequently obtained sequences belonged to the nc10 phylum (ettwig et al. 2009 ). alternatively, it could be that the wwtps screened in this study harbored nc10 bacteria that have more mismatches with the primers used. these primers are based on a limited amount of nc10 sequences presently known, and therefore could miss members of this phylum which have a (slightly) different nucleotide composition at the primer positions. m. oxyfera-type bacteria were not detected in the wwtp in varsseveld, which may be caused by severe mismatches of the primers or the prevailing conditions in this system. varsseveld is the only plant in our selection that makes use of a membrane bio reactor (mbr). before the biological treatment starts in this mbr, the wastewater is filtered thoroughly. furthermore, to prevent the membranes from clogging, intensive aeration and periodical chemical cleaning are necessary. this might cause an environment not favorable for nc10 bacteria. sludge from the treatment plant in lieshout was selected to enrich a nitrite-dependent methane oxidizing culture. after 308 and 315 days of enrichment in the 3-l bioreactor, experiments to determine the conversion rates of methane and nitrite were performed. the observed stoichiometry for methane to nitrite was 3 ch 4 :9.8 no 2 − (308 days) and 3 ch 4 :10.1 no 2 − (315 days), which is comparable to the theoretical value 3 ch 4 :8 no 2 − (raghoebarsing et al. 2006; ettwig et al. 2009 ). the slight deviation indicated that there was still additional denitrification. this process may have been using other electron donors than methane like organic compounds or ammonium. similar observations were made in previous studies (raghoebarsing et al. 2006; hu et al. 2009 ). the measured conversion rates for methane to nitrite in this study are relatively low compared to previous experiments (raghoebarsing et al. 2006; ettwig et al. 2008 ettwig et al. , 2009 . it is possible that the protein content in the current enrichment was overestimated since organic compounds of activated sludge interfered with the bca assay (ras et al. 2008) . organic compounds might be present in the lieshout enrichment, as a remainder of the uasb process. to detect m. oxyfera-type bacteria on a functional level, primers targeting the pmoa gene were used on samples originating from the wwtp lieshout and the enrichment culture. the new pmoa primer a189_b (luesken et al. 2011 ) was combined with the widely applied 682r primer (holmes et al. 1995) . using this combination, pmoa sequences were found that did not cluster with m. oxyfera but clustered with m. capsulatus (gammaproteobacteria) or c. polyspora (gammaproteobacteria). this is in accordance with previous results where it was shown that m. oxyfera has some critical mismatches with, especially, the known pmoa reverse primers (luesken et al. 2011) . with a nested pcr approach, sequences clustering with the pmoa sequence present in the genome of m. oxyfera were retrieved in all tested samples. the sequences detected in the inoculum and in the wwtp 1 year after taking the inoculum were similar to each other and to the sequence of the m. oxyfera pmoa gene. this may indicate that there is a small but persistent population of nc10 bacteria present in this wwtp. two different pmoa primer combinations were used in a direct pcr on samples of the enrichment originating from the wwtp lieshout. after 332 days of enrichment, pmoa sequences could be retrieved. these results implied that when m. oxyfera-type bacteria were enriched, a direct pcr with specific pmoa primers can be used. conclusively, we showed that nine out of ten selected wwtps harbor small amounts of m. oxyfera bacteria clustering within the nc10 phylum, using molecular tools for screening and detection of these bacteria. in addition, an enrichment culture (60-70%) from wastewater sludge was obtained performing nitrite-dependent methane oxidation at ambient temperature. the m. oxyfera-type bacteria present in this enrichment were identified and monitored using specific pmoa primers. these data suggested that biomass from wwtp systems could be a potential source to start pilot scale reactors with very efficient biomass retention. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. anaerobic and complementary treatment of domestic sewage in regions with hot climates-a review combination of 16s rrna-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations phylogenetic identification and in-situ detection of individual microbial-cells without cultivation mitigation of global greenhouse gas emissions from waste: conclusions and strategies from the intergovernmental panel on climate change (ipcc) fourth assessment report. working group iii (mitigation) greenhouse gas production: a comparison between aerobic and anaerobic wastewater treatment technology denitrifying bacteria anaerobically oxidize methane in the absence of archaea enrichment and molecular detection of denitrifying methanotrophic bacteria of the nc10 phylum nitrite-driven anaerobic methane oxidation by oxygenic bacteria evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related enrichment of denitrifying anaerobic methane oxidizing microorganisms combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: nitrosococcus mobilis and nitrospira-like bacteria as dominant populations adaptation of a freshwater anammox population to high salinity wastewater anaerobic treatment: a key technology for a sustainable management of wastes in europe use of the upflow sludge blanket (usb) reactor concept for biological wastewater-treatment, especially for anaerobic treatment pmoa primers for detection of anaerobic methanotrophs a microbial consortium couples anaerobic methane oxidation to denitrification protein extraction from activated sludge: an analytical approach the sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms mega4: molecular evolutionary genetics analysis (mega) software version 4.0 anaerobic digestion. i. microbiology of anaerobic digestion the sharon((r))-anammox((r)) process for treatment of ammonium rich wastewater a new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium candidatus 'methylomirabilis oxyfera key: cord-012062-xlw92xoe authors: ichinose, hitomi; yoshida, makoto; fujimoto, zui; kaneko, satoshi title: characterization of a modular enzyme of exo-1,5-α-l-arabinofuranosidase and arabinan binding module from streptomyces avermitilis nbrc14893 date: 2008-09-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1551-x sha: doc_id: 12062 cord_uid: xlw92xoe a gene encoding an α-l-arabinofuranosidase, designated saaraf43a, was cloned from streptomyces avermitilis. the deduced amino acid sequence implies a modular structure consisting of an n-terminal glycoside hydrolase family 43 module and a c-terminal family 42 carbohydrate-binding module (cbm42). the recombinant enzyme showed optimal activity at ph 6.0 and 45°c and was stable over the ph range of 5.0–6.5 at 30°c. the enzyme hydrolyzed p-nitrophenol (pnp)-α-l-arabinofuranoside but did not hydrolyze pnp-α-l-arabinopyranoside, pnp-β-d-xylopyranoside, or pnp-β-d-galactopyranoside. debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-o-α-l-arabinofuranosyl-α-l-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-o-α-l-arabinofuranosyl-α-l-arabinofuranoside and methyl 3-o-α-l-arabinofuranosyl-α-l-arabinofuranoside were not. these data suggested that the enzyme only cleaves α-1,5-linked arabinofuranosyl linkages. the analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. these results indicate that the enzyme is definitely an exo-1,5-α-l-arabinofuranosidase. the c-terminal cbm42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the cbm42 bound to branched arabinofuranosyl residues. removal of the module decreased the activity of the enzyme with regard to debranched arabinan. the cbm42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains. l-arabinose residues are widely distributed in plant cell walls, where they are present in polymers such as arabinans, arabinoxylans, arabinogalactans, and arabinogalactan proteins (carpita and gibeaut 1993) . plant cell walls are becoming important because worldwide attention has now focused on bioethanol production to combat global warming and to safeguard global energy. because of competition between food and fuel production, lignocellulose is expected to be utilized for fuel ethanol production in the future. generally, lignocellulose is composed of almost 40% cellulose together with about 20% hemicelluloses which mainly consist of pentoses such as xylose and arabinose (saha 2003) . hemicelluloses are often detrimental to the production of bio-ethanol because pentoses are less efficiently converted to ethanol than are hexoses (aristidou and penttilä 2000; skoog and hahn-hägerdal 1988) . in contrast, l-arabinose has potential as a useful sugar in the food industry. the sugar has a sweet taste but is not readily absorbed by the body (seri et al. 1996) . l-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (seri et al. 1996; sanai et al. 1997; osaki et al. 2001) . thus, l-arabinose may be useful in preventing excess sucrose utilization. because the structure of l-arabinose-containing polysaccharides are highly variable and complex, a wide variety of α-l-arabinofuranosidases (ec 3.2.1.55) which have various substrate specificities are necessary for the hydrolysis of such polysaccharides and the production of l-arabinose. we have previously purified some α-l-arabinofuranosidases and elucidated their substrate specificities towards structurally defined substrates (kaneko et al. 1993; kaneko et al. 1994; kaneko et al. 1998a; kaneko et al. 1998b; kaneko et al. 1998c; kaneko et al. 1998d; kawabata et al. 1995; kusakabe et al. 1975; matsuo et al. 2000; yang et al. 2006; yoshida et al. 1994) . the substrate specificities of the α-l-arabinofuranosidases studied were very broad; however, one of the enzymes (α-l-arabinofuranosidase ii) from streptomyces chartreusis (matsuo et al. 2000) has strict substrate specificity. this enzyme hydrolyzed only the α-1,5-linkages of linear arabinan and arabinooligosaccharides in an exo-acting manner (matsuo et al. 2000) . the amino acid sequence of the enzyme indicated that this enzyme belongs to family 43 (gh43) of the glycoside hydrolase family (http://www.cazy. org/cazy/). because the substrate specificity and the amino acid sequence were quite distinct from known α-l-arabinofuranosidases, it represents a novel type of enzyme as an exo-1,5-α-l-arabinofuranosidase (matsuo et al. 2000) . the gh43 family incorporates a wide variety of enzyme activities including β-xylosidase (ec 3.2.1.37), α-l-arabinofuranosidase (exo-1,5-α-l-arabinofuanosidase), bifunctional β-xylosidase/α-l-arabinofuranosidase, endo-arabinanase (ec 3.2.1.99), β-xylanase (ec 3.2.1.8), and exo-β-1,3galactanase (ec 3.2.1.145). therefore, a detailed functional characterization of α-l-arabinofuranosidase ii using the recombinant enzyme with its mutants would be interesting because all the enzymes belonging to gh43 have the same holding and catalysis in the same inverting mechanism even though the enzyme activities are different. we have attempted heterologous expression of α-l-arabinofuranosidase ii from s. chartreusis, but unfortunately, the effort was unsuccessful. when the amino acid sequence of exo-1,5-α-l-arabinofuranosidase from s. chartreusis was subjected to a blast search, a similar gene from streptomyces avermitilis (sav1043) was identified. the gene designated saaraf43a gene encodes not only a gh43 catalytic domain similar to α-l-arabinofuranosidase ii from s. chartreusis but also the family 42 carbohydrate-binding module (cbm42) at the cterminus. in the present study, the gene was cloned from s. avermitilis, and the recombinant protein expressed in escherichia coli was characterized. substrates p-nitrophenyl α-l-arabinofuranoside (pnp-α-l-araf), p-nitrophenyl α-l-arabinopyranoside (pnp-α-l-arap), p-nitrophenyl β-d-galactopyranoside (pnp-β-d-galp), p-nitrophenyl β-dxylopyranoside (pnp-β-d-xylp) and larch wood arabinogalactan, oat spelts xylan, and birchwood xylan were purchased from sigma chemical company (st. louis, mo, usa). debranched arabinan, wheat arabinoxylan, and arabinopentaose were obtained from megazyme international (wicklow, ireland). gum arabic was obtained from nacali tesque (kyoto, japan). nihon syokuhin kakoh (fuji, japan) supplied corn hull arabinoxylan. sugar-beet arabinan was prepared according to methods described elsewhere (kusakabe et al. 1975) . methyl 2-o-, methyl 3-o-, and methyl 5-o-α-larabinofuranosyl-α-l-arabinofuranosides (arabinofuranobiosides) were synthesized as described previously . β-1,3-galactan was prepared as previously reported (ichinose et al. 2005) . the oligonucleotide primers were designed from the terminal of position 1318577-1319938 on the s. avermitilis genome sequence (on the website at http://avermitilis.ls.kitasato-u.ac. jp) for ligation into the ndei-hindiii site of the expression vector pet30 (novagen, madison, wi, usa). the 5′-primer (5′-aacatatgaccgccccggcctcgccctc-3′) contained two adenine residues to prevent removal of the terminal nucleotide by the exonuclease activity of highfidelity dna polymerase, the ndei site (shown in italics) and a stretch of 20 nucleotide residues encoding the nterminal amino acids of mature saaraf43a. the 3′-primer (5′-aaaagcttttctgcgtagaacgtggcgtcc-3′) contained two adenine residues, the hindiii site (shown in italics) and 22 nucleotide residues encoding c-terminal amino acids of the mature saaraf43a. for amplification of the catalytic domain of saaraf43a, a pair of primers was designed as follows. the 5′-primer (5′-aacata tgaccgccccggcctcgccctc-3′) contained the ndei site (shown in italics) and a stretch of 20 nucleotide residues encoding the n-terminal amino acids of mature saaraf43a. the 3′-primer (5′-aaaagcttatcagccactggaa taccga -3′) contained two adenine residues, the hindiii site (shown in italics) and 20 nucleotide residues encoding c-terminal amino acids of the catalytic domain of saar-af43a. polymerase chain reactions (pcrs) were performed with the pair of primers described above using kod-plus-dna polymerase (toyobo, osaka, japan). in the reactions, genomic dna from s. avermitilis was used as a template. the amplified products were subcloned into pgem-t easy vector (promega, madison, wi, usa) followed by sequencing analysis. the target genes were digested with ndei and hindiii and ligated into the corresponding restriction site of the pet30 vector. e. coli strain bl21 gold (de3) (stratagene, la jolla, ca, usa) was transformed by the resultant plasmids. the transformants were grown in 100 ml of luria-bertani medium at 37°c until they had reached an optimal optical density of 0.2 at 600 nm; expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 24 h at 25°c. the cells were harvested and then resuspended in 5 ml of 50 mm phosphate buffer (ph 7.2) followed by sonication for 5 min. after centrifugation to remove insoluble material, the supernatant was used as the crude enzyme which was purified on a ni-nitrilotriacetic acid (nta) agarose (qiagen gmbh, hilden, germany) column (5×50 mm). the eluted enzyme was applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), and the relevant fractions were pooled and dialyzed against deionized water. the final preparation thus obtained was used as the purified enzyme. the enzyme assay mixture contained 25 μl 2 mm pnp-α-l-araf, 20 μl mcilvaine buffer, ph 6.0 (0.2 m na 2 hpo 4 / 0.1 m citric acid) and 5 μl of the enzyme solution. the reactions were carried out at 45°c for 10 min and were terminated by the addition of 50 μl of 0.2 m na 2 co 3 . the amount of pnp released was determined at 400 nm with an extinction coefficient of 19,608 m −1 cm −1 . one unit of enzyme activity is defined as the amount of enzyme that released 1 μmol of pnp per minute from pnp-α-l-araf under these conditions. the protein concentration was determined using the bca protein assay kit (pierce, rockford, il, usa) with bovine serum albumin as the standard. the effects of ph on the activity and stability of saaraf43a were examined in a series of mcilvaine buffers ranging in ph from 3.0 to 8.0 and in atkins-pantin buffers (0.2 m boric acid/0.2 m kcl/0.2 m na 2 co 3 ) ranging from ph 8.0 to 10.0. the activities were assayed under the conditions described for the standard method. in order to determine the effect of ph on enzyme stability, the enzymes were preincubated at various ph values in the absence of substrate at 30°c for 60 min, and the residual activity was then assayed using the standard method. the effects of temperature on the activity of α-l-arabinofuranosidase were determined from 4 to 50°c. with the exception of temperature, the assay conditions were the same as described for the standard method. for the temperaturestability measurements, the enzymes were pre-incubated at various temperatures at ph 6.0 for 60 min, and the residual activity was determined using the standard method. to determine the substrate specificities, various pnpglycosides were used as substrates. in these cases, the assay method was identical to that described for pnp-α-l-araf. for polysaccharide and arabinofuranobioside substrates, a solution was prepared containing 5 μl of enzyme solution (0.0015 unit), 20 μl mcilvaine buffer (ph 6.0) and 25 μl 0.5% (w/v) substrate (β-1,3-galactan, gum arabic, larch arabinogalactan, debranched arabinan, arabinan, oat spelt xylan, birchwood xylan, wheat arabinoxylan, corn hull arabinoxylan, or arabinofuranobiosides). after incubation for the appropriate reaction time at 30°c, the reaction was stopped by boiling for 5 min. analysis of the hydrolysis products of arabinofuranobiosides was carried out by determining the concentration of l-arabinose released by the enzyme using the somogyi-nelson method (somogyi 1952) . for the hydrolysis products analysis of debranched arabinan, the enzyme (75 micro units) was incubated with 0.25% (w/v) the substrate at 30°c. the reaction mixtures were subjected to high-performance anion-exchange chromatography and identified using a pulsed amperometric detection (hpaec-pad) system. the samples were analyzed using a carbopac™ pa1 column (4×250 mm, dionex, sunnyvale, ca, usa) and elution with 0.1 m naoh (0-5 min), followed by a linear gradient (5-35 min) of sodium acetate (0-0.2 m) at a flow rate of 1 ml/min. for the hydrolysis of arabinopentaose, 25 μl of 0.5% (w/v) arabinopentaose was incubated with 5 μl of the enzyme solution (75 micro units) in the reaction mixture. after incubation for the appropriate reaction time at 30°c, the reaction was stopped by boiling for 5 min. the hydrolysis products were analyzed by hpaec-pad analysis as described above. the catalytic activities of the enzymes with and without cbm42 towards pnp-α-l-araf and debranched arabinan were compared as follows. the enzyme assay mixture contained 25 μl 2 mm pnp-α-l-araf, 20 μl mcilvaine buffer, ph 6.0, and 5 μl of 1 μm enzyme solution. the reactions were carried out at 20°c and for periods of up to 20 min. the amount of pnp released was determined at 400 nm with an extinction coefficient of 19,608 m −1 cm −1 . for debranched arabinan, a solution was prepared containing 5 μl of 1 μm enzyme solution, 20 μl mcilvaine buffer (ph 6.0), and 25 μl of 0.5% (w/v) substrate. after incubation for the appropriate reaction time at 20°c, the reaction was stopped by boiling for 5 min. for the hydrolysis products of debranched arabinan, the concentration of l-arabinose released by hydrolysis from debranched arabinan was determined using the somogyi-nelson method (somogyi 1952) . the affinity of saaraf43a for a range of soluble polysaccharides was determined by affinity gel electrophoresis. the method was essentially as described previously (takeo 1984) , with the exception of the buffer system, and used arabinoxylan from wheat, arabinogalactan from larch, arabinan from sugar beet pulp, and debranched arabinan as the polysaccharides. the separating gels were made up of 7% (w/v) acrylamide in 0.375 m tris (ph 9.4), and the stacking gels consisted of 2.2% (w/v) acrylamide in 0.125 m tris (ph 6.8). for the separating gels containing ligands, polysaccharide was added to gel mixtures at concentrations ranging from 0.0005% to 0.2% prior to polymerization. the proteins (5 μg) were electrophoresed at 10 ma/gel for 3 h at 4°c. the cathode buffer (ph 8.9) contained 0.042 m tris and 0.045 m glycine, and the anode buffer (ph 8.0) was prepared with 0.12 m tris. sds-page standard low range (bio-rad, hercules, ca) was used as a negative, non-interacting control. proteins were stained with coomassie brilliant blue r-250. quantitative assessment of binding was carried out as described in the previous report . nucleotide and amino acid sequences of saaraf43a and expression of recombinant protein using the amino acid sequence of α-l-arabinofuranosidase ii from s. chartreusis, a gene from s. avermitilis (genbank accession number bac68753) encoding a putative protein (sav1043, annotated as a putative secreted α-l-arabinofuranosidase ii in the ncbi database) was found. the dna sequence was 1,443 bp long and putatively encodes a 481amino acid protein. the deduced amino acid sequence was compared with sequences in the protein database using blast (on a national center for biotechnology information website, http://www.ncbi.nlm.nih.gov/blast/). the sequence (residues 28 to 481), saaraf43a, resembled the following sequences, in order of decreasing similarity: putative α-arabinofuranosidase ii from stigmatella aurantiaca dw4/3-1 (68% identity and 82% similarity, genbank accession number, eau68983.1); clostridium cellulosome enzyme, dockerin type i: glycoside hydrolase, family 43: α-l-arabinofuranosidase b from clostridium thermocellum atcc 27405 (62% identity and 79% similarity, genbank accession number, eam44536.1); α-arabinofuranosidase ii from s. chartreusis [60% identity and 74% similarity, dna data bank of japan (ddbj) accession number, baa90772.1]; hypothetical protein bh1867 from bacillus halodurans c-125 (59% identity and 74% similarity, ddbj accession number, bab05586.1); hypothetical protein from neurospora crassa or74a (58% identity and 74% similarity, genbank accession number, eaa29844.1); and hypothetical protein chgg_03607 from chaetomium globosum cbs 148.51 (55% identity and 70% similarity, genbank accession number, eaq91672.1). the sequence at the n-terminus (amino acids 1 to 27) was predicted to be a signal sequence (sosui[http://bp.nuap.nagoya-u.ac.jp/ sosui/]), and residues 28 to 315 of saaraf43a showed sequence similarity to the gh43 enzymes, while residues 349 to 481 showed similarity with the cbm42 (fig. 1a,b) . to confirm that saaraf43a is an exo-1,5-α-l-arabinofuranosidase, the saaraf43a gene was expressed and then characterized. the dna fragments encoding the mature region of saaraf43a and the catalytic domain of saaraf43a (the cbm42-deleted mutant) were amplified by pcr and then cloned into the expression vector pet30, respectively. the recombinant saaraf43a was successfully produced in e. coli bl21(de3) at a level of approximately 280 mg/l after the induction for 24 h at 25°c. the recombinant protein was purified by a ni-nta column chromatography and gave a single band on sds-page when visualized by staining with cbb r-250 (fig. 2, lane 1) . the cbm42-deleted mutant also could be expressed in e. coli in its active form (fig. 2, lane 2) . the molecular mass of both recombinant saaraf43a and the cbm42-deleted mutant as estimated from sds-page were found to be 53 and 35 kda, respectively. these corresponded to their predicted molecular mass (52 and 33 kda). when recombinant saaraf43a was incubated with pnp-α-l-arabinofuranoside, α-l-arabinofuranosidase activity was detected, and the specific activity was 2.92 u/mg. this value is similar to that of α-l-arabinofuranosidase ii from s. chartreusis (3.16 u/mg in matsuo et al. 2000) . the enzyme showed maximal activity at ph 6.0 and was stable between ph 5.0 and 6.5, under which conditions more than 80% of the activity was retained (data not shown). the enzyme achieved maximal activity at 45°c, and more than 80% activity was retained after incubation at 30°c for 60 min (data not shown). to determine the enzyme activities included in the gh43 family, saaraf43a was incubated with appropriate substrates (β-1,3-galactan, gum arabic, larch arabinogalactan, debranched arabinan, arabinan, oat spelt xylan, birchwood xylan, wheat arabinoxylan, corn hull arabinoxylan, or pnp-glycosides). the enzyme only showed significant activity toward pnp-α-l-arabinofuranoside among tested pnp-glycosides. when the enzyme was incubated with polysaccharides, saaraf43a only had significant activity on debranched arabinan which mainly consists of α-1,5-linked arabinofuranosyl residues among the polysaccharides tested. therefore, the detailed substrate specificities of the enzyme were characterized by using various types of structurally defined arabinofuranooligosaccharides. the degrees of the hydrolysis of three kinds of regioisomer of arabinofuranobiosides by saar-af43a was monitored over time (fig. 3) . the enzyme hydrolyzed methyl α-l-1,5-arabinofuranobioside to arab-inose and methyl α-l-arabinofuranoside but did not hydrolyze the 1,2-and 1,3-linkages (fig. 3) . those data suggest that saaraf43a discriminates between the different types of linkage and that the enzyme is specific for 1,5-linked α-arabinofuranosyl residues. the hydrolysis products of α-1,5-arabinofuranopentaose and debranched arabinan were analyzed by hpaec-pad (fig. 4, 5) . when saaraf43a was incubated with α-1,5arabinofuranopentaose (fig. 4) , l-arabinose and arabinotetraose were detected as the initial hydrolysis products (after 7 min incubation), and the levels of shorter oligosaccharides such as arabinotriose and arabinobiose then gradually increased as the period of incubation progressed. in contrast, only arabinose was released from debranched arabinan (fig. 5) . these results indicate that the enzyme released arabinofuranose from the substrate in an exo-type mode of action. thus, saaraf43a is definitely an exo-1,5α-l-arabinofuranosidase. to determine sacbm42 binding to polysaccharides, affinity gel electrophoresis was performed (fig. 6) . sacbm42 showed affinity for arabinan and wheat arabinoxylan but did not bind to arabinogalactan. interestingly, sacbm42 did not bind to debranched arabinan even though it is a good substrate for saaraf43a. the k d value for arabinan (0.023 mg/ml) was slightly smaller than that for arabinoxylan (0.075 mg/ml). in contrast, the cbm42-deleted mutant did not show any affinity for all substrates tested (data not shown). to understand the role of sacbm42 in the activity of saaraf43a, the cbm42-deleted mutant of saaraf43a was constructed. the mutant showed almost the same activity as the wild-type with regard to pnp-α-l-araf, but there was a approximately 1 μg of each sample was separated on 12% polyacrylamide gel significant reduction in the hydrolysis rate when the mutant was incubated with debranched arabinan (fig. 7) . the data suggest that sacbm42 binds to small amount of arabinosyl side chains that were left on debranched arabinan and thus increases the enzyme concentration around the substrate. gh43 includes several kinds of α-l-arabinanases. the crystal structure of α-l-arabinanases from cellvibrio japonicus (cjarb43a) and bacillus subtilis (bsarb43a) have been determined (nurizzo et al. 2002; proctor et al. 2005) . bsarb43a is a typical endo-type of enzyme which randomly hydrolyzes linear 1,5-arabinan, while cjarb43a is exo-acting and releases arabinotriose from the 1,5arabinan. therefore, the amino acid sequences of sa-araf43a and α-l-arabinofuranosidase ii from s. chartreusis were compared with those of cjarb43a and bsarb43a (fig. 1a) . the most obvious sequential difference in saaraf43a (exo-1,5-α-l-arabinofuranosidase) with bsarb43a (endo-arabinanase) is the presence of three loop insertions (indicated as l4, l6, and l9 in fig. 1a ) and six loop deletions (indicated as l1, l2, l3, l5, l7, and l8 in fig. 1a) . the five-residue loop (l1) which is relating to the endo-mode of action in bsarb43a (proctor et al. 2005) was not observed in saaraf43a, while the large loop insertion (l6) that seals the putative aglycon end of the substrate binding groove in cjarb43a (proctor et al. 2005) was also present in the exo-1,5-α-l-arabinofuranosidases. the important acidic residues for the catalysis such as e221 (catalytic acid), d38 (catalytic base), d158 (function unknown) in cjarb43a which were demonstrated by the mutagenesis study (nurizzo et al. 2002) were completely conserved in saaraf43a (fig. 1a) . in contrast, the amino acids which were shown to be important in substrate binding in cjarb43a (nurizzo et al. 2002; proctor et al. 2005) were not completely conserved in saaraf43a. the w94 in cjarb43a was conserved, but phenylalanines such as f114 and f176 in cjarb43a were not conserved, suggesting that the manner in which exo-1,5-α-l-arabinofuranosidase binds the substrate is different from that of the arabinanases. the substrate recognition mechanism of exo-1,5-α-l-arabinofuranosidase will be different from the other α-l-arabinofuranosidases. crystal structures of α-l-arabinofuranosidases belonging to gh51 and gh54 suggest that these α-larabinofuranosidases have an active site pocket which is suitable to recognize monosaccharide (hövel et al. 2003; taylor et al. 2006; miyanaga et al. 2004) . the substrate specificities of these enzymes are rather broad so that these enzymes do not recognize aglycon side so strictly. in contrast, exo-1,5-α-l-arabinofuranosidase specifically cleaves the internal α-1,5-linkage of two arabinofuranosyl residues. therefore, the enzyme would possess at least two subsites capable to bind α-1,5-linked arabinofuranosyl residues. in general, the arabinans consist of a linear chain of α-1,5-linked arabinofuranosyl residues that may be alternately substituted at the o-3 and/or o-2 with additional arabinofuranosyl residues. therefore, exo-1,5-α-l-arabinofuranosidase could not hydrolyze sugar beet arabinan by the steric hindrance caused by 1,3-and/or 1,2-linked α-arabinofuranosyl side chains at the subsite + 1 or −1. on the other hand, cbm42 was first found associated to gh54 α-l-arabinofuranosidase from trichoderma reesei as a xylan-binding module (nogawa et al. 1999) . the crystal structure of aspergillus kawachii gh54 α-l-arabinofuranosidase was resolved, and it was demonstrated that the cbm in the c-terminal of the gh54 enzyme binds to arabinose (miyanaga et al. 2004 ). miyanaga et al. (2006) characterized the cbm in detail and found that cbm42 binds to arabinose side chains in arabinoxylan and arabinan. cbm42 consists of three similar repeated peptides of about 40 residues, each referred to as subdomains α, β, and γ, and these three units assemble around the pseudo threefold axis to form a globular structure. this combination of three subdomains results in a fold similar to the "β-trefoil fold". this fold is common to cbm13 and certain plant galactosebinding lectins such as ricin b-chain and abrin b-chain (boraston et al. 2004 ). the residues involved in arabinosebinding in akcbm42 which were demonstrated in the akcbm42/arabinose complex (miyanaga et al. 2004) showed that most of the residues are strictly conserved among all three subdomains of sacbm42 (fig. 1b) . in subdomain β, aromatic amino acid in akcbm42 (y417) stacking with arabinose was replaced by lys in sacbm42 (k382). however, subdomain β of sacbm42 would be able to bind to the substrate because the other residues involved in substrate binding were conserved. actually, the properties of sacbm42 was quite similar to akcbm42. generally, the α-l-arabinofuranosidases belonging to gh54 preferentially remove arabinosyl side chains from arabinan. therefore, the properties of cbm42 which specifically bind the arabinofuranose side chains of hemicelluloses would be suitable for the substrate specificity of the catalytic module. in contrast, exo-1,5-α-l-arabinofuranosidase hydrolyzes debranched arabinan (linear 1,5-arabinan) and does not cleave the arabinosyl side chains of arabinan. synergism in arabinan hydrolysis would be expected if gh54 α-larabinofuranosidase and saaraf43a were present together. however, no gh54 enzymes were found in genome database of s. avermitilis. therefore, it would be interesting to determine whether sacbm42 enhances the activity of saaraf43a. to elucidate this, the cbm42-deleted mutant of saaraf43a was constructed, and the activity of the mutant for debranched arabinan was tested (fig. 7) . the activity of saaraf43a was significantly higher than the cbm42-deleted mutant, suggesting that the binding ability of sacbm42 for only small amounts of arabinosyl side chains remaining in debranched arabinan increases the enzyme concentration around the substrate. the debranched arabinan had been obtained by treating the arabinan with arabinofuranosidase, which removed the 1,3-and 1,2linked α-arabinofuranosyl residues. the methylation analysis of arabinan and enzyme-digested arabinan revealed that 32-45% of side chains were left on debranched arabinan (kaneko et al. 1998a, c, d) , indicating the possibility of sacbm42 to bind the debranched arabinan. in order to obtain more detailed information about this unique function, structure-function analysis of sacbm42 would be required. the open reading frame (orf) sav1043 lies adjacent to orf sav1044 and forms an operon in the s. avermitilis genome. the expressions of both genes were regulated by the same promoter. blast search revealed that the orf sav1044 might encode an α-l-arabinofuranosidase which would have different substrate specificity from sav1043. in this study, we showed the orf sav1043 encode an exo-1,5-α-l-arabinofuranosidase. the proximity of the orfs may suggest that the actinomycete has advantage for the hydrolysis of polysaccharides including arabinose. in conclusion, we demonstrated in this work that saaraf43a is definitely an exo-1,5-α-l-arabinofuranosidase. saaraf43a is a modular enzyme possessing cbm42 at its c terminus. we also demonstrated that sacbm42 binds to the arabinofuranosyl side chains of arabinan and arabinoxylan. interestingly, it plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains. saaraf43a provides a good model for investigating the hydrolytic activity of the modular enzyme. in the future, we hope to provide additional information that should be of use in further determinations of the structure-function relationships of saaraf43a. metabolic engineering applications to renewable resource utilization x4 modules represent a new family of carbohydratebinding modules that display novel properties carbohydrate-binding modules: fine-tuning polysaccharide recognition structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth crystal structure and snapshots along the reaction pathway of a family 51 α-l-arabinofuranosidase an exo-β-1,3-galactanase having a novel β-1,3-galactan binding module from phanerochaete chrysosporium substrate specificity of α-l-arabinofuranosidase from bacillus subtilis 3-6 toward arabinofuranooligosaccharides purification and some properties of intracellular α-l-arabinofuranosidase from aspergillus niger 5-16 purification and some properties of α-l-arabinofuranosidase from bacillus subtilis 3 the core trisaccharide of α-l-arabinofuranan: synthesis of methyl 3,5-di-o-α-l-arabinofuranosyl-α-l-arabinofuranoside substrate specificity of α-l-arabinofuranosidase from trichoderma reesei substrate specificity of α-l-arabinofuranosidase from streptomyces diastatochromogenes 065 toward arabinose-containing oligosaccharides purification and substrate specificities of two α-larabinofuranosidases from aspergillus awamori ifo 4033 substrate specificities of α-l-arabinofuranosidases produced from two species of aspergillus niger synthesis of regioisomeric methyl α-l-arabinofuranobiosides some properties of araban degrading enzymes produced by microorganisms and enzymatic preparation of arabinose from sugar beet pulp purification, characterization and gene cloning of two α-larabinofuranosidases from streptomyces chartreusis gs901 crystal structure of a family 54 α-l-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose the family 42 carbohydrate-binding module of family 54 α-l-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose an alpha-l-arabinofuranosidase from trichoderma reesei containing a noncatalytic xylan-binding domain cellvibrio japonicus α-l-arabinanase 43a has a novel five-blade βpropeller fold l-arabinose feeding prevents increases due to dietary sucrose in lipogenic enzymes and triacylglycerol levels in rats tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of cellvibrio japonicus arabinanase 43a hemicellulose bioconversion inhibition of sucrose digestion and absorption by l-arabinose in rats l-arabinose selectively inhibits intestinal sucrase in an uncompetitive manner and suppresses glycemic response after sucrose ingestion in animals xylose fermentation notes on sugar determination affinity electrophoresis: principles and applications structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, araf51, from clostridium thermocellum synergy between an α-l-arabinofuranosidase from aspergillus oryzae and an endo-arabinanase from streptomyces coelicolor for degradation of arabinan detection of α-l-arabinofuranosidase activity in isoelectric focused gels using 6-bromo-2-naphtyl α-l-arabinofuranoside acknowledgment this work was supported financially by a grant-inaid (development of biomass utilization technologies for revitalizing rural areas) from the ministry of agriculture, forestry and fisheries of japan.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-257136-zpeh8pmc authors: huang, xin; chen, jianing; yao, gang; guo, qingyong; wang, jinquan; liu, guangliang title: a taqman-probe-based multiplex real-time rt-qpcr for simultaneous detection of porcine enteric coronaviruses date: 2019-04-26 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-09835-7 sha: doc_id: 257136 cord_uid: zpeh8pmc swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. to develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (tgev) n, porcine epidemic diarrhea virus (pedv) m, porcine deltacoronavirus (pdcov) m, and porcine enteric alphacoronavirus (peav) n genes respectively. a taqman-probe-based multiplex real-time rt-qpcr assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. the results showed that the limit of detection can reach as low as 10 copies in singular real-time rt-qpcr assays and 100 copies in multiplex real-time rt-qpcr assay, with all correlation coefficients (r(2)) at above 0.99, and the amplification efficiency at between 90 and 120%. this multiplex real-time rt-qpcr assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. the multiplex real-time rt-qpcr assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. the results manifested that tgev and pdcov were the main pathogens in these samples, accompanied by co-infections. this well-established multiplex real-time rt-qpcr assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses. electronic supplementary material: the online version of this article (10.1007/s00253-019-09835-7) contains supplementary material, which is available to authorized users. porcine enteric viruses, the pathogens of viral diarrhea in pigs, caused huge economic losses in the swine industry in recent years ). there were more than ten enteric viruses discovered from swine gut in the past, including but not limited to transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus within these swine enteric viruses, coronaviruses are the most devastating pathogens responsible for acute diarrhea, vomiting, dehydration, and high mortality in neonatal and suckling piglets. according to the genetic and antigenic characters, coronavirus was divided into four genera: alpha-, beta-, gamma-, and delta-cov (woo et al. 2010) . during the past years, some alphacoronaviruses (pedv, tgev, and peav) and a deltacoronavirus (pdcov) emerged or reemerged in the pig farms and resulted in severe diarrhea and mortality in the early stage of suckling piglets. the tgev and pedv were the traditional causal agents responsible for diarrhea in pigs for the past decades. however, the variant strains of pedv were discovered since 2010 and circulating in pig herds thereafter, showing up to 100% death rate for piglets younger than 1-week-old (lyoo et al. 2017; sun et al. 2012) . another swine enteric virus, porcine deltacoronavirus (pdcov), was firstly discovered from healthy pig herds by a electronic supplementary material the online version of this article (https://doi.org/10.1007/s00253-019-09835-7) contains supplementary material, which is available to authorized users. research team of hong kong in 2012 when they did a molecular epidemiology study in 3137 mammals and 3519 birds (woo et al. 2012) . two years later, this virus was reported to cause severe diarrhea and/or vomiting and atrophic enteritis in the usa ) and later on in china (song et al. 2015) . subsequently, a novel porcine enteric alphacoronavirus peav was discovered in 2017 from some pig farms located in southern china, causing more than 24,000 piglets' death (gong et al. 2017) . this virus was also named as seacov (pan et al. 2017) or sads-cov (zhou et al. 2018b ) by the other research groups. all these four swine enteric coronaviruses, causing similar clinical symptoms and pathological changes in piglets, circulate in the pig herds and result in huge economic losses across the world in recent years. to develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, a taqman-probe-based multiplex real-time rt-qpcr assay was established to simultaneously detect tgev, pedv, peav, and pdcov from the same reaction vial. to our knowledge, this is the first multiplex real-time rt-qpcr method for swine enteric coronaviruses detection. the tgev was previously propagated and preserved in our laboratory. the pedv and pdcov were isolated from clinical samples and confirmed by conventional pcr and dna sequencing (genewiz, suzhou, china). the nucleoprotein gene of peav (genbank access number: mf370205) was synthesized from genewiz biotech company (suzhou, china). field samples were collected from diarrheal piglets between 2015 and 2018 from liaoning, shandong, chongqing, shaanxi, ningxia, and gansu provinces. all samples were stored at − 80°c until use. the primers and taqman probes for real-time qpcr assay were designed by the software beacon designer 7 (premier biosoft international, palo alto, ca, usa). the detailed information of primers and probes were listed in table 1 . all clinical samples were resuspended with phosphatebuffer saline (pbs), vortexed and centrifuged at 12,000×g at 4°c for 10 min. an aliquot of 250 μl supernatant was applied for total rna extraction with rnaiso reagent (takara, dalian, china) following the manufacturer's instruction. the total rna in rnasefree water was reversely transcribed into cdna using revertaid first strand cdna synthesis kit (thermo scientific, waltham, usa). the cdna was subjected to real-time qpcr analysis with the multiplex rt-qpcr method established in this study. the m genes of pedvand pdcov, and n genes of tgevand peav were constructed into pet-30a(+) vector. the recombinant plasmids were linearized by smai enzyme digestion and recovered by pcr purification kit. the purified recombinant plasmids were quantified by spectrophotometric analysis. the copy number of recombination plasmids was calculated by using the following formula (huang et al. 2009 ): to establish the standard curves for single coronavirus, each plasmid was diluted in a tenfold series, from 10 7 copies/μl to 10 1 copies/μl. for multiplex standard curves, each of the four linearization plasmids was adjusted to 4 × 10 9 copies/μl and pooled with equal volume to made 1 × 10 9 copies/μl of each plasmid. the pooled plasmid was then diluted serially by tenfold to establish multiplex standard curves. all real-time qpcr reaction systems were set to a volume of 20 μl. for single qpcr amplifying tgev, pedv, and pdcov, 10 μl 2 × transstart probe qpcr supermix (transgene, beijing, china), 200 nm primers and probe each, 1 μl plasmid dna template, and 6.8 μl nuclease-free water were pooled and mixed. for peav amplification, 10 μl 2 × transstart probe qpcr supermix (transgene, beijing, china), 500 nm each primer, 100 nm probe, 1 μl plasmid dna template, and 6.8 μl nuclease-free water were pooled and mixed. all reactions were amplified on a bio-rad cfx96™ real-time system (bio-rad, hercules, ca, usa) at 94°c for 30s, followed by 40 cycles of 94°c for 5 s and 60°c for 30s. for reaction system of multiplex real-time pcr, 10 μl 2 × transstart probe qpcr supermix combined with all primers, probes, templates, and nuclease-free water to a final volume of 20 μl. the concentrations of each primer and probe of pedv, pdcov, tgev, and peav were optimized for better outputs. the amplifying cycles of multiplex qpcr were carried out as same as singular real-time qpcr. the cq value higher than 35 was considered negative. all qpcr results were analyzed by cfx manager™ software. to analyze the sensitivity of established multiplex rt-qpcr, the linearization standard plasmids prepared above were diluted tenfold serially to a final concentration between 1.1 × 10 7 copies/μl and 1.1 × 10 1 copies/μl in nuclease-free water. the diluted standard plasmids were used as templates for realtime qpcr amplification. to estimate the specificity of this established multiplex rt-qpcr, standard dnas, or cdnas of major swine viruses, including porcine astrovirus (pastv), porcine kobuvirus (pkv), type o foot-and-mouth disease virus (fmdv-o), type a foot-and-mouth disease virus (fmdv-a), porcine reproductive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), porcine rotavirus (prv), porcine circovirus 2(pcv2), and pseudorabies virus (prv) were used as templates for amplification. the nuclease-free water was served as negative template control. to evaluate its repeatability, tenfold serially diluted standard template between 1.1 × 10 7 copies/μl to 1.1 × 10 1 copies/μl were used to test the coefficients of variation of real-time pcr. for intra-assay repeatability, all samples were triplicated. for inter-assay repeatability, the assays were repeated three times individually at different locations. a total of 354 fecal samples were collected from diarrheal pig farms located in liaoning, shandong, chongqing, shaanxi, ningxia, and gansu provinces of china between 2015 and 2018. all samples were diluted fivefold with sterile phosphate-buffered saline (pbs), vortexed, and centrifuged at 1847×g at 4°c for 20 min. the supernatant was collected and used to extract viral rna with trizol reagent (invitrogen, carlsbad, ca, usa). the cdnas were generated by reverse transcript system (promega, madison, wi, usa) using extracted total rna as templates and hexamer random primers. all cdna from clinical samples were measured by the multiplex rt-qpcr assay developed in this study. to develop a multiplex real-time rt-qpcr, the single real-time rt-qpcrs for detection of the individual virus were firstly established with different fluorescence-labeled target probes, for details, fam for pedv m gene, cy5 for pdcov m gene, hex for tgev n gene, and texasred for peav n gene. the standard curves for each virus were generated using 1.1 × 10 7 copies to 1.1 × 10 1 copies of tenfold serially diluted linearized plasmids conceiving target genes. the results demonstrated that the single real-time rt-qpcr assays for each virus were successfully established at the limit of detection at approximately 10 copies (fig. 1 ). all the standard curves showed an excellent correlation coefficient and amplification efficacy, for instance, pedv (r 2 = 1; eff% = 95.7), pdcov (r 2 = 1; eff% = 100.9), tgev (r 2 = 0.999; eff% = 106.0), and peav (r 2 = 0.995; eff% = 98.9), indicating that the single real-time rt-qpcr for each virus was valid and reliable. to establish the multiplex real-time rt-qpcr method, all primer sets, probes, and serially diluted standard plasmids for detection of pedv, pdcov, tgev, and peav were mixed with 2 × transstart probe qpcr supermix and nuclease-free water. the concentrations of each primer and probe were optimized for the optimum output. the optimal final concentrations of primers and probes were as follows: 300 nm primer and 100 nm probe for pedv, 200 nm primer and 200 nm probe for pdcov, 500 nm primer and 100 nm probe for tgev, and 500 nm primer and c the amplification curves (top) and a standard curve (bottom) for detection of tgev n gene were generated. the probe was labeled with hex at 5′-end and bhq1 at 3′-end. d the amplification curves (top) and a standard curve (bottom) for detection of pedv n gene were generated. the probe was labeled with texasred at 5′-end and bhq2 at 3′-end 50 nm probe for peav. the results demonstrated that the multiplex real-time rt-qpcr could detect all target genes of these four viruses efficiently with high correlation values (fig. 2) . all the standard curves showed excellent correlation coefficient and amplification efficacy, for details, pedv (r 2 = 0.999; eff% = 103.5), pdcov (r 2 = 0.999; eff% = 104.7), tgev (r 2 = 0.999; eff% = 105.6), and peav (r 2 = 1; eff% = 100.4) (fig. 2) . the limit of detection of this multiplex rt-qpcr was approximately 100 copies of each virus per reaction (fig. 2) . the specificity of multiplex real-time rt-qpcr assay to evaluate the specificity of the multiplex rt-qpcr developed in this study for swine enteric coronavirus detection, the dnas/cdnas of nine other major swine viruses were used as templates for amplification with this multiplex system. the cdnas of pedv, pdcov, tgev, and peav were served as positive control while the nuclease-free water was used as a negative control. the results displayed that all swine enteric coronaviruses were successfully detected. however, there was no positive signal detected from other nine swine viruses, pastv, pkv, fmdv-o, fmdv-a, prrsv, csfv, prv, pcv2, prv, and negative control using this multiplex rt-qpcr system (fig. 3) , demonstrating that the taqmanprobe-based multiplex rt-qpcr system was highly specific. to determine the sensitivity of this multiplex real-time rt-qpcr, tenfold serial dilution of linearized plasmid mixtures were added to the amplification system. the results showed that 1.1 × 10 2 copies of tgev, pedv, pdcov, and peav were detectable with the ct values at 28.75, 28.94, 30.03, and 28.4 respectively ( table 2 ). however, 1.1 × 10 1 copies of each target were not detectable in the same amplification system (table 2) . also, the amplification exhibited reliable and high efficacy, pedv (r 2 = 0.998; eff% = 96.04), pdcov (r 2 = 0.992; eff% = 91.38), tgev (r 2 = 0.998; eff% = 94.57), and peav (r 2 = 0.999; eff% = 98.60). this multiplex real-time rt-qpcr demonstrated the same sensitivity level when using viral rnas as templates (supplemental fig. s1 ). these results implied that the taqman fig. 2 establishment of multiplex real-time rt-qpcr. a amplification curves and b standard curves of optimized multiplex taqman-probe-based realtime rt-qpcr for detection of pedv, pdcov, tgev, and peav were generated at the optimum amplification conditions. the correlation coefficient and amplification efficacy of the standards curves, pedv (r 2 = 0.999; eff% = 103.5), pdcov (r 2 = 0.999; eff% = 104.7), tgev (r 2 = 0.999; eff% = 105.6), and peav (r 2 = 1; eff% = 100.4), were ideal for detecting the target genes enteric coronaviruses (pedv, tgev, pdcov, and peav) as less as 10 to 100 copies. to estimate the reproducibility of the multiplex real-time pcr, tenfold serial dilution of pooled linearization plasmids were in a triplicate manner for both intra-assay and inter-assay. for intra-assay, the standard plasmids were amplified three times simultaneously. for inter-assay, standard curves were done at three individual times and using a different batch of a standard substance. as shown in table 3 , the coefficient of variation for pedv intra-assay was 0.30-1.56% and interassay was 0.44-1.79%. the pdcov amplification exhibited the coefficients of variation at 0.09-5.51% and 1.91-3.78% for inter-assay and intra-assay respectively. as for tgev, the coefficients of variation were 0.33-1.29% and 0.95-3.70% corresponding to intra-assay and inter-assay. the amplification of peav showed relative higher coefficients of variation, which was 0.39-7.32% for intra-assay and 1.08-4.52% for inter-assay. these results indicated that the taqman-probe-based multiplex real-time rt-qpcr assay established in this study was repeatable and reliable. the cdnas originated from 354 clinical samples were subjected to amplification by this multiplex real-time rt-qpcr and confirmed by conventional rt-pcr (supplemental fig. s2 ). the results demonstrated that the samples from henan and shaanxi provinces were swine enteric coronavirusnegative while the gansu province suffered a high prevalence of coronaviruses, showing 83.9% (26/31) tgev, 48.4% (15/ 31) pdcov, and 38.7% (12/31) pedv positive (table 4 ). in ningxia province, tgev was also highly prevalent but the positive rates for the other three swine enteric coronaviruses were quite low or not detectable (table 4 ). only pedv (15/ 62, 24.2%) and pdcov (8/62, 12.9%) among these four coronaviruses were detectable from clinical samples of chongqing. all these four swine enteric coronaviruses were detectable in samples of liaoning province, with tgev positive rate at 12.6% (18/143) and the other three were less than 10% (table 4) . overall, pdcov (121/354, 34.2%) and tgev (72/354, 20.3%) were the main coronaviruses detected from all these clinical samples. the positive rate for the newly emerged coronavirus in guangdong, china, peav, was rarely found from these provinces. we further analyzed the co-infection of coronaviruses for all these positive samples. the results elucidated that the main co-infection were caused by a dual coronavirus, for example, 13 pdcov and tgev co-infection samples, five pedv and pdcov co-infection samples, and four pedv and tgev co-infection samples (fig. 4) . some clinical samples were co-infected by three (9 pedv, pdcov, and tgev co-infection) or all four coronaviruses (fig. 4) . these results indicated the pathogens of viral diarrhea disease in chinese pig farms were complicated. porcine diarrheal disease is one of the most severe diseases in pig farms. swine enteric coronaviruses are the most significant pathogens causing diarrhea in piglets, especially for newborn piglets (butler et al. 2014 ). all swine enteric coronaviruses, including traditional pedv, tgev, and newly emerged pdcov, peav, could cause serious diarrhea, vomiting, dehydration, weight loss, and up to 100% death in suckling piglets (gong et al. 2017; hsu et al. 2018) . previously, some investigators developed a serial of elisa assays to detect pedv, tgev, or pdcov infections based on m, n proteins or whole virus (luo et al. 2017; ma et al. 2016; su et al. 2016 ). however, molecular diagnostic tools, rather than serological methods, for swine enteric coronaviruses are urgently needed due to their similar and high pathogenicity to suckling piglets, lacking a mature immune system. for swine enteric coronavirus detection, conventional rt-pcr, multiplex rt-qpcr, or pan-coronaviruses rt-pcr methods targeting m, n, s, or polymerase genes were developed in recent years (hsu et al. 2018; song et al. 2006) . additionally, zhou et al. (2018a) recently reported a specific real-time pcr for the detection of peav. to our knowledge, there was no such method that could simultaneously detect and differentiate tgev, pedv, pdcov, and peav from the same detection vial. to solve this urgent and important issue, we developed this multiplex real-time rt-qpcr for the detection and differential diagnosis of the swine enteric coronaviruses circulating in pig herds. taqman-probe-based real-time qpcr is a rapid, high specificity, high sensitivity, and great reproducibility detection tool for identification of viruses (chang et al. 2014 ). among the seven open reading frames (orfs) and four structural protein genes, s (spike), e (envelope), m (membrane), n (nucleocapsid) of coronaviruses (su et al. 2018 ), the m and n genes are highly conserved within the same antigenic group but less homologous between each of these four swine enteric coronaviruses (yang et al. 2019) . to establish a high-specific multiplex real-time rt-qpcr for the detection and differential diagnosis of swine enteric coronaviruses, primer sets, and taqman probes were designed targeting the highly conserved regions of pedv or pdcov m genes and tgev or peav n genes, based on bioinformatics analysis of each virus. our results demonstrated that each primer and probe set can only detect target gene itself and could not bind with any other targets, indicating high specificity. our results showed that the singular real-time rt-qpcrs were capable to detect as less as 10 copies of pedv, pdcov, tgev, and peav templates. however, the detection limit of each target gene in the multiplex real-time rt-qpcr was approximately 100 copies, implying that the sensitivity of multiplex real-time rt-qpcr was lower than that of singular realtime rt-qpcr probably due to the competition between primers, probes, templates, and reagents. the analysis of clinical diarrhea samples using this developed multiplex rt-qpcr elucidated that the co-infection of swine enteric coronaviruses commonly existed in some pig farms. co-infection of swine enteric coronaviruses may cause recombination between co-infected viruses. during 2012 and 2016, some new swine enteric coronaviruses were generated by recombination with pedv and tgev and spread across the central eastern european countries (akimkin et al. 2016; belsham et al. 2016; boniotti et al. 2016) . the recombination might create more virulent enteric virus strains or new viruses, leading to potential outbreaks or pandemics of swine viral diarrhea. additionally, co-infections may promote the evolution of non-pathogenic enteric viruses into high virulent and pathogenic viruses. a recent example showed the existence of pdcov in pig herds for many years in china mainland and hong kong but without observed clinical symptoms (pan et al. 2017; woo et al. 2012) . unfortunately, this virus caused severe outbreaks in some states of the usa since 2014 (ma et al. 2015; wang et al. 2014 ). this virulence change was most probably caused by the previous exposure to pedv or other co-infected swine enteric viruses. therefore, swine enteric coronaviruses co-infection may bring risks for the prevention and control of swine diarrhea. in summary, we developed a taqman-probe-based multiplex real-time rt-qpcr with high specificity and sensitivity for simultaneous detection and differential diagnosis of swine enteric coronaviruses, pedv, tgev, pdcov, and peav. this real-time rt-qpcr assay is of great significance for the prevention and control and epidemiological investigation of swine viral diarrhea. conflict of interest the authors declare that they have no competing interests. ethical statement this article does not contain any studies with human participants or animals performed by any of the authors. new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic the application of a duplex reverse transcription real-time pcr for the surveillance of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 a new bat-hku2-like coronavirus in swine detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in taiwan development of a reverse transcription multiplex real-time pcr for the detection and genotyping of classical swine fever virus development and application of a recombinant m protein-based indirect elisa for the detection of porcine deltacoronavirus igg antibodies development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virus origin, evolution, and virulence of porcine deltacoronaviruses in the united states two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus discovery of a novel swine enteric alphacoronavirus (seacov) in southern china multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis a recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus a novel duplex taqman probebased real-time rt-qpcr for detecting and differentiating classical and variant porcine epidemic diarrhea viruses outbreak of porcine epidemic diarrhea in suckling piglets detection and genetic characterization of deltacoronavirus in pigs coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus generation, identification and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid evaluation of two singleplex reverse transcription-insulated isothermal pcr tests and a duplex real-time rt-pcr test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus development of a taqman-based real-time rt-pcr assay for the detection of sads-cov associated with severe diarrhea disease in pigs fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-012430-3uvhoca9 authors: sanchis, joaquin; fernández, layla; carballeira, j. daniel; drone, jullien; gumulya, yosephine; höbenreich, horst; kahakeaw, daniel; kille, sabrina; lohmer, renate; peyralans, jérôme j.-p.; podtetenieff, john; prasad, shreenath; soni, pankaj; taglieber, andreas; wu, sheng; zilly, felipe e.; reetz, manfred t. title: improved pcr method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates date: 2008-11-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1678-9 sha: doc_id: 12430 cord_uid: 3uvhoca9 saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. traditional protocols of whole plasmid amplification such as stratagene’s quikchange™ sometimes fail when the templates are difficult to amplify. in order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (pcr) method which constitutes an improvement over existing protocols. in the first stage of the pcr, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. in the second stage, the amplified sequence is used as a megaprimer. sites composed of one or more residues can be randomized in a single pcr reaction, irrespective of their location in the gene sequence.the method has been applied to several enzymes successfully, including p450-bm3 from bacillus megaterium, the lipases from pseudomonas aeruginosa and candida antarctica and the epoxide hydrolase from aspergillus niger. here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. comparison of the results with the performances of previous protocols reveals the efficiency of the improved method. directed evolution constitutes a powerful method for engineering essentially any property of an enzyme, including thermostability, substrate acceptance, and enantioselectivity, as documented by recent reviews (arndt and müller 2007; arnold and georgiou 2003a; brakmann and schwienhorst 2004; hibbert et al. 2005; rubin-pitel and zhao 2006; reetz 2006; bershtein and tawfik 2008) . it is based on the appropriate combination of gene mutagenesis and expression coupled with high-throughput screening or selection. a variety of mutagenesis techniques have been devised, the most often used methods being error-prone polymerase chain reaction (eppcr; leung et al. 1989 ; cadwell and joyce the screening effort (reymond 2005) can be reduced significantly because this is generally the bottleneck of directed evolution. to this end, we have proposed and implemented iterative saturation mutagenesis (ism) as a general approach which comes closer to the above goal (reetz et al. 2006b; reetz et al. 2006c; . the concept of ism can be used to influence very different catalytic properties of an enzyme, such as thermostability in the form of b-fit (reetz et al. 2006b; or substrate acceptance and enantioselectivity using casting (reetz et al. 2006c ; bartsch et al. 2008; liang et al. 2007 ). based on structural information of the enzyme and by focusing on positions expected to be crucial for a given catalytic property, several sites, composed of one or more residues, can be selected and randomized. saturation mutagenesis can be performed by applying a variety of different molecular biological methods developed during the past two decades (arndt and müller 2007; dominy and andrews 2003; georgescu et al. 2003; hogrefe et al. 2002; kirsch and joly 1998; zheng et al. 2004; reetz 2004) . currently, the most popular approach is the use of the stratagene quikchange™ protocol (hogrefe et al. 2002) . the original form of quikchange™ is limited to the introduction of only one mutation at a given amino acid position. moreover, problems related to the primer length and design also persist. different approaches have been reported which overcome the problem of primer design by using partially overlapped (zheng et al. 2004) or even nonoverlapping oligonucleotides (kirsch and joly 1998) , where the resulting amplicon is used as a megaprimer (sarkar and sommer 1990; miyazaki and takenouchi 2002) , thereby completing the synthesis of the plasmid in a second pcr. however, we encountered difficulties in the application of these methods to certain recalcitrant targets such as plasmids containing p450-bm3 from bacillus megaterium and pseudomonas aeruginosa lipase a genes. in such cases, the amplification and the subsequent introduction of mutations proved to be unsuccessful. given the importance of saturation mutagenesis in enzyme evolution (reetz 2004; reetz 2006) , any substantial simplification and/or extension of existing protocols would be of great interest. here, we present an improved method to perform saturation mutagenesis in the gene of difficult-to-amplify templates using a single two-stage whole-plasmid pcr. in the first stage of the pcr, two mutagenic primers or a mutagenic primer and an antiprimer (a non-mutagenic (silent) primer used to complete the complementary extension as well as to help in opening and uncoiling the dna) are used for only a few cycles which are needed to generate the megaprimer. once the megaprimer is generated, the second stage begins in which the annealing temperature is increased to eliminate priming by the oligonucleotide primers, and a further 20 cycles are carried out to amplify the mutated plasmid ( fig. 1) . this procedure is an extension of the method proposed by kirsch and joly, modified to work not only on the complicated templates mentioned above but also on any template as an alternative to standard techniques and especially in cases where quikchange™ fails. recently, tseng et al. (2008) published a note on related work, also based on the work of kirsch and joly, while we were finalizing our experiments. our findings broadly agree with their results, but our method probes further into the possible uses of this approach, as in saturation mutagenesis at multiple sites and more importantly into the quality of the resulting libraries. we demonstrate that optimal primer localization and orientation are essential to increase the yield in the amplification of these difficult sequences. additionally, this method intrinsically avoids problems arising from palindromes, hairpins or self-pairing in oligonucleotides that plague other methods based on overlapping primers. the gene is represented in blue, the vector backbone in gray, and the formed megaprimer in black. in the first stage of the pcr, both the mutagenic primer (positions randomized represented by a red square) and the antiprimer (or another mutagenic primer, shown to the right) anneal to the template and the amplified sequence is used as a megaprimer in the second stage. finally, the template plasmids are digested using dpni, and the resulting library is transformed in bacteria. the scheme to the left of the figure illustrates the three possible options in the choice of the megaprimer size for a single site randomization experiment. the scheme to the right represents an experiment with two sites simultaneously randomized plasmids petm11-p450-bm3 (8474 bp), pucpcl6an (6994 bp; jaeger et al. 1997) , petm11-calb (6282 bp), and pqe-aneh (4631 bp; cedrone et al. 2003) were used as templates to create saturation mutagenesis libraries. the p450-bm3 gene from b. megaterium (narhi and fulco 1982) was pcr amplified from genomic dna (atcc 14581/strain no. 32 at dsmz gmbh, germany) and cloned into the expression vector petm11 (embl vector collection, germany) using the ncoi and saci restriction sites. the calb gene from candida antarctica was pcr amplified from genomic dna (atcc 32657 at dsmz gmbh, germany) and cloned into the expression vector petm11 (embl vector collection, germany) using the ncoi and noti restriction sites. all degenerate oligonucleotides (see table 1 for more details) were synthesized by invitrogen (germany). pcr amplifications were carried out with kod hot start dna polymerase (novagen, usa) and digested with dpni (new england biolabs, uk). the sequencing was performed on plasmid dna extracted from pooled colonies using qiaprep miniprep kit (qiagen, germany). all transformations were carried out with the same batch of homemade escherichia coli dh5α chemocompetent cells according to a standard protocol. 1 kb dna ladder was obtained from fermentas. the reactions were performed in a total volume of 50 μl, and the reaction mixtures were prepared according to the quikchange™ protocol, using 50 ng of template. pcr conditions were: initial denaturation 3 min at 95°c followed by 16 cycles of 1 min at 95°c, 1 min of annealing at the required temperature (t m -5°c), 1 min/kb of extension at 72°c; and 2 min/kb of final extension (table 1 ). to remove template plasmid, pcr amplified mixtures were digested with dpni (20 u, 10× dpni buffer) for 1 h at 37°c, after which another aliquot of dpni (20 u) was added and digestion continued for 1 h more. a 2-μl aliquot was used to transform e. coli dh5α. as p. aeruginosa lacks the dam methylation system (stover et al. 2000) , the pcr template used in this protocol had to be obtained from e. coli in order to be recognized by dpni. the method reported by kirsch and joly (1998) was applied as described by the authors. this called for 50 ng of template to be mixed with 12.5 pmol of each oligonucleotide in a 50-μl reaction. the amplification program was as follows: initial denaturation 3 min at 95°c followed by 9 cycles of 30 s at 95°c, annealing 1 min at 55°c, extension 1 min at 68°c, with a further 9 cycles of 30 s at 95°c, extension 6 min at 68°c; and final extension 16 min at 68°c. pcr-amplified reaction mixtures were digested with dpni and transformed in e. coli as described above. the method reported by zheng et al. (2004) was carried out using 50 ng of template and 12.5 pmol of each oligonucleotide in a 50 μl reaction. pcr cycle conditions were: initial denaturation 3 min at 94°c followed by 16 cycles of 1 min at 94°c, annealing 1 min at 52°c, extension 1 min/kb at 68°c; and final extension 1 h at 68°c. dpni digestion and transformation were performed as described above. improved method of the present study the sequence of the antiprimers was designed to amplify megaprimers of different lengths: small, with the antiprimer annealing site close to the open reading frame; medium, with the annealing site that gives a megaprimer encompassing half length of the plasmid; and large, with the annealing site giving the whole plasmid minus a small part of the gene. the sequence of each antiprimer was adjusted to have an identical or similar t m value as the mutagenic oligonucleotide used to produce the library. the reactions were performed in a final volume of 50 μl containing 50-100 ng of template and 5 pmol of each primer. the amplification program was as follows: initial denaturation 3 min at 95°c (for p. aeruginosa lipa template, 5 min at 98°c) followed by 5 cycles of 30 s at 95°c, annealing 1 min at 55°c (this parameter depend on the t m of the oligonucleotides used, and in all cases a gradient pcr was performed), extension 1 min/kb according to the megaprimer size at 72°c. the second stage consisted of 20 cycles of 30 s at 95°c and extension at 1 min/kb of template at 68°c; and a final extension of 2 min/kb of template at 68°c. for each reaction, dpni digestion and transformation were performed as described above. experiments with the p450-bm3 gene here, we use our method for the introduction of 1-and 2codon mutations in the p450-bm3 gene cloned in petm11. we first applied it to the site saturation mutagenesis of position f87 (one codon, nnk randomization; table 2 ). the yields of the amplification were analyzed by agarose gel electrophoresis (fig. 2 ). the quikchange™ protocol gave an average of 80 colonies (see experimental procedures in "materials and methods" for details). this result was 3.5-fold better than both the kirsch and joly method and the zheng et al. method (28 and 23 colonies, respectively). the application of our method gave significantly different results with respect to the size of the generated megaprimer. in the above case, the medium-sized megaprimer gave results comparable to quikchange™ with 71 colonies. the small-sized megaprimer yielded 168 colonies, which are 2.5-fold better. ultimately, the generation of the large megaprimer gave the best results with more than the randomization frequencies at position f87 were assessed for each protocol. the inserts harbored the desired nnk randomization in that position. the statistical distribution of the different nucleotides, assuming complete randomization, should have been 25% of each nucleotide for the first two positions and 50% (only t and g are expected) for the third nucleotide in the codon. however, the observed distribution was different with respect to the protocol used (table 3) . when quikchange™ was used for f87 saturation, the wild-type ttt codon was only partially randomized since it turned out that all three deoxythymidines were highly conserved (from 67% to 83% conservation for individual nucleotides). when the kirsch and joly protocol was used, the native codon was less conserved (from 60% to 72% conservation for individual nucleotides). with our method, the randomization was more efficient (large megaprimer), reaching around 50% for the whole codon. we further extended our method to the simultaneous randomization of two codons corresponding to positions m185 and l188 in p450-bm3. in this part of the study, we compared our improved method with the zheng et al. method (table 2) . around 40 colonies were obtained on average with the zheng et al. protocol. our method yielded 15 colonies with the small-and 37 colonies with the medium-sized megaprimers. furthermore, the generation of the large megaprimer gave 245 colonies after transformation. this result demonstrates that even for randomization at two positions, our method is 6.5-fold more efficient than the previously published protocol by zheng et al. sequencing performed on the plasmid dna confirmed that the wild-type codons at positions m185 and l188 were substituted by two nnk randomized codons. as with position f87, similar randomization efficiency was obtained for the simultaneous saturation of m185 and l188 where the zheng et al. protocol generated conservation rates from 39% to 79% for individual nucleotides, while our method (large megaprimer) gave rates ranging from 39% to 53% for individual nucleotides (table 4 ). the latter results demonstrate that this method never underperformed the existing mutagenesis strategies and is more efficient in almost every case. site-saturation mutagenesis was also performed at two adjacent positions in the same oligonucleotide in the lipa gene of p. aeruginosa pao1 (accession number x63390). the yields of amplification were analyzed by agarose gel electrophoresis (fig. 3) . one oligonucleotide for the library and different antiprimers annealing outside the cloning site were used (fig. 4) . the three amplification reactions were each carried out with the two possible directionalities. firstly, three pcr reactions were performed using the forward mutagenic primer and a different reverse antiprimer in each one to produce megaprimers, (small, medium, and large size, respectively) clockwise (cw) with respect to lipa gene (fig. 4a) . secondly, we used the reverse complementary primers: reverse mutagenic primer and forward antiprimers to synthesize the anticlockwise (acw) megaprimers (small, medium, and large) with respect to lipa gene (fig. 4b) . the results from these experiments were compared with other protocols described for site-directed and saturation mutagenesis ( table 5 ). the standard quikchange™ and the kirsch and joly methods failed to amplify the plasmid, and no colonies were found after transformations. implementation of the zheng et al. method to this template yielded only five colonies. on the other hand, the yields from the application of our method were extremely variable depending on the size and position of the megaprimers. the small megaprimers were successfully amplified in both directions in spite of the differences in gc content between these two occurrence frequencies were estimated using the relative signal amplitudes at each position from sequencing chromatogram. the statistical distribution of the different nucleotides, assuming complete randomization, should be 25% of each nucleotide for the first two positions and 50% (only t and g are expected) for the third nucleotide in the codon. the sequencing was performed using a pool of all the obtained transformants in each experiment. wild type nucleotide occurrences are emphasized with bold type (table 5 , fig. 3: lanes 1 and 4) . nevertheless, no colonies were obtained after transformation with the small acw megaprimer and only seven in the case of small cw megaprimer, showing that the whole plasmid pcr was not completely accomplished. the yield was slightly increased when the medium and large megaprimers were produced in the anticlockwise direction, giving almost the same number of colonies as the zheng et al. method. however, it was with the medium-sized clockwise megaprimer that we achieved the best result with 277 colonies: an improvement of more than 55-fold with respect to the zheng et al. method. the quality of these libraries was determined by sequencing the pcr products before transformation: they showed the expected degeneracy at the mutated positions. experiments with the candida antarctica lipase b and aspergillus niger epoxide hydrolase genes in order to further demonstrate the broad applicability of this method, we introduced site-directed mutations and saturation mutagenesis in two different templates. the first example is the application of our method to c. antarctica lipase b, cloned in petm11 plasmid. here, we attempted to perform site-saturation mutagenesis at two distant positions simultaneously in a single pcr. these two positions, separated by 171 bp, were successfully randomized giving a high quality saturation mutagenesis library (data not shown) and yielding an average of 400 colonies after transformation of dpni digested reactions. the second example utilizes the gene of a. niger epoxide hydrolase cloned in the pqe vector (cedrone et al. 2003) . our improved method was successfully applied in the case of site-directed mutagenesis or saturation mutagenesis in regions where the restricted possibilities of primer design given by quikchange™ force the inclusion of sequences that contain palindromes or overlapping regions (oligonucleotides bwt-f, bwt-r, pri b2-r, pri b3-r; table 6 ). the problem was easily solved by the selection of a reverse antiprimer outside of the gene (sp1-f), in an area free of those inconveniences, with a similar t m as the forward mutagenic primer. the application of our method resulted in a high percentage of the expected mutants and insertions were never observed (table 6 ). for the negative control, no primers were added and no colonies were observed in the selection plates after dpni digestion. the number of colonies represents the average of three independent experiments. quikchange™ method has been tried with several pcr enhancers and in many different conditions with the same negative results. a gc content of the formed megaprimer quikchange™ and our improved method applied to introduce site-directed mutagenesis in the gene of a. niger epoxide hydrolase cloned in the pqe vector (pqe-aneh plasmid, mutant xm10). although pqe-aneh is easily amplified by quikchange™, mutations in this specific target region are difficult to achieve due to the presence of palindromes or repetitions in its sequence. the problem is solved by using im (see main text). qc quikchange™, im improved method, bwt-f, bwt-r, pri b2-r, pri b3-r mutagenic primers, sp1 silent helper primer (for sequences see table 1 ), tr. number of obtained transformants, seq. results of the sequences of the three first randomly chosen colonies, d deletion of nucleotides inside or around the mutated region, i insertion of nucleotides inside or around the mutated region the previously reported method of kirsch and joly (1998) is essentially a combination of quikchange™ (stratagene, la jolla, ca, usa) and megaprimer (sarkar and sommer 1990) leading to one-step pcr mutagenesis. in our extension of this protocol, we applied saturation mutagenesis at specific residues using modified oligonucleotide concentrations and annealing cycles in order to increase the yield of the final product. furthermore, we compared different annealing positions of the helper oligonucleotide-called the antiprimer-which can be silent or mutagenic, obtaining different megaprimer sizes. during the first stage of pcr, the megaprimer is generated by amplifying the region between the two oligonucleotide primers. contrary to what was reported by kirsch and joly (same number of cycles per stage), we suggest a drastic reduction in the number of cycles during the first stage. when we have tried the kirsch and joly method in our systems, overproduction of megaprimer was observed in the exponential stage. as a result, in the second or linear stage, megaprimers self anneal leading to unproductive complexes. a reduced amount of oligonucleotides (5 pmol) in the pcr reaction, an increased amount of template and only 3 to 5 cycles, enhance the yield of the final plasmid library (fig. 5) . we applied our method in several practical examples where existing protocols (namely quikchange™, kirsch and joly as well as zheng et al.) failed to generate saturation mutagenesis libraries. we discuss them here separately. experiments with the p450-bm3 gene the optimization of p450-bm3 is an intensive field of research in protein engineering as recently reviewed by tee and schwaneberg (2007) . to the best of our knowledge, two methods are currently used for the creation of bm3 saturation mutagenesis libraries, namely overlap extension pcr and quikchange™ (peters et al. 2003; arnold and georgiou 2003b; wong et al. 2008; li et al. 2008) . overlap extension pcr is a time-consuming protocol that often yields a low number of colonies which is not well suited for the creation of libraries. moreover, in our hands, applying the standard quikchange™ protocol to p450-bm3 gene failed in almost all cases (no amplification, insertions or deletions were observed, data not shown). a major problem in applying saturation mutagenesis concerns those cases in which large plasmids are involved. indeed, p450-bm3 was cloned in the vector petm11, resulting in a large construct of 8474 bp. for the construction of the f87 library of p450-bm3, our method gave equal or better results than quikchange™, the kirsch and joly method, and the zheng et al. protocol with respect to both quality (randomization) and quantity (number of colonies). the size of the megaprimer has a considerable impact on the results. in this particular case, the large megaprimer was the most successful for library creation. the ideal methodology for performing site saturation mutagenesis is the one yielding (1) a uniform statistical distribution of the desired mutations (quality criteria) and (2) a number of colonies sufficient to get 95% coverage of the given library in one step (quantity criteria). accordingly, our method is one step closer to the ideal site saturation mutagenesis protocol which allowed us to construct two different saturation libraries for p450-bm3, in less time, with the greatest number of transformants and with the highest level of randomization. experiments with p. aeruginosa lipase a gene different techniques such as overlap extension pcr and/or subcloning of a mutated dna fragment have been applied to mutate certain residues by saturation mutagenesis or sitedirected mutagenesis in p. aeruginosa lipase a gene (liebeton et al. 2000; reetz et al. 2001; liebeton et al. 2001; fuji et al. 2005; reetz et al. 2006a; ). whole-plasmid pcr, quikchange™, and other methods have proven to be rapid and highly efficient methods to introduce these mutations directly in the plasmid in only one step. however, these protocols failed when applied to p. aeruginosa lipase a constructs, giving very poor yields or no amplification. it is known that high gc containing templates cause particular problems such as inconsistent amplification or none at all even with the addition of enhancers or the use of different polymerases. thus, we analyzed the gc content of this template revealing that the region encompassing the lipa gene (coding for lipase a) and the liph gene (coding for the foldase) has a gc content of 68% compared to the overall value of 54%. to address this problem, we again applied our method, placing the antiprimer at three positions and with two directionalities, resulting in six fig. 5 recommended working conditions. the amount of template and oligonucleotides are given for 50 μl of reaction mixture. the figure also depicts the three possible choices in the election of the antiprimer position megaprimers as described above. in this case, we hypothesize that the megaprimer might aid in the amplification of the gc rich regions by wedging the dna strands open, thereby facilitating extension. from the results of our experiments (fig. 3, table 5 ), it appears that the combination of size and possibly gc content in the megaprimer are responsible for the efficient amplification of the whole plasmid. although the medium sized megaprimers (clockwise and anticlockwise) amplified fragments of almost identical length, those fragment contained different parts of the template having different gc content. the anticlockwise megaprimer mainly encompasses vector dna with lower gc content and leads to unsuccessful amplification probably for the same reason as in the other methods, i.e., the polymerase cannot replicate the remaining region (with high gc content). on the other hand, the clockwise megaprimer, which includes the lipa and liph genes (with high gc content) as well as part of the vector, yields a successful amplification. this seems to confirm the hypothesis that opening the gc rich region with a large dna fragment (megaprimer) aids the polymerase in accomplishing the whole plasmid pcr. the oligonucleotides giving the small sized clockwise megaprimer were positioned in the lipa and liph genes. although the gc content in this region of the plasmid is very high (68%), the megaprimer was successfully amplified; however, it is possible that the size of the megaprimer was insufficient to keep the plasmid open during the second stage of the pcr. the large anticlockwise megaprimer was not properly amplified during the first stage of the pcr (fig. 4) where the main product was an indistinct band. due to the large size of this megaprimer and its orientation, the polymerase is probably facing the same problems that plague the standard methods: a long fragment to amplify, primed from within a region of high gc content. to summarize, the orientation and the annealing positions of the primers used to amplify the medium size clockwise megaprimer created conditions favorable to the polymerase for amplification of the plasmid. this megaprimer which encompasses a large portion of the high gc area efficiently disrupted this region of the template, facilitating whole plasmid amplification in the second stage of the pcr. experiments with c. antarctica lipase b and a. niger epoxide hydrolase genes our protocol was also applied to templates that could be effectively mutated by the quikchange™ protocol but where the use of the improved method represents an advantage or is able to overcome some problems derived from the sequence of the template. we illustrate this with two examples: c. antarctica lipase b cloned in petm11 and a. niger epoxide hydrolase cloned in pqe60. the advantage gained in the case of c. antarctica lipase b was the achievement of simultaneous randomization at two distant sites in one pcr. with the quikchange™ method, the same result would have required two pcrs and four mutagenic primers. therefore, using accessible laboratory materials, our method saves time and costs, representing an alternative to high-priced kits as, e.g., quikchange™ (stratagene) multi-site directed mutagenesis and quik-change™ xl that depend on special enzyme mixtures and tailor-made solutions. in the case of a. niger epoxide hydrolase, we wanted to perform site-directed mutagenesis in a problematic region of the gene. one of the most limiting features of quikchange™ relates to the primer design when performing site-directed mutagenesis or saturation mutagenesis in regions containing repetitions or palindromes. in these regions, the formation of primer-dimers is facilitated, frequently leading to multiple insertions of the primer sequence or less often to deletions of one or more nucleotides. due to the small difference in the size of these erroneously mutated plasmids with respect to the original template, it is very difficult to distinguish and isolate them from the desired mutant by gel electrophoresis, thereby contaminating and decreasing the quality of the library and consequently increasing the screening effort in directed evolution. the fact that quikchange™ requires the use of complementary forward and reverse primers can make it impossible to overcome the problem of primer-dimers. because our method uses an antiprimer located outside of the target (palindrome containing) region, we were able to circumvent the problem of primer dimers. additionally, the flexibility in the choice of the antiprimer allows a sequence matching the t m of the mutagenic primer to be designed, all of which leads to successful amplification of palindromic targets as illustrated by the experiments with a. niger epoxide hydrolase (table 6 ). in conclusion, the modified and extended protocol we present offers high applicability and rapidity in performing saturation mutagenesis. it avoids traditional subcloning steps and requires only one randomized oligonucleotide per library plus an antiprimer (non-mutagenic oligonucleotide) which can be used repeatedly in different saturation mutagenesis reactions-in contrast to quikchange™, which requires two primers per mutation. this antiprimer can be designed in an area that avoids palindromes, hairpins, or overlapping regions with the mutagenic primer. additionally, as hplc-purified oligonucleotide primers are highly recommended for generating saturation mutagenesis libraries, the repeated use of one specific antiprimer for each template is very cost effective. we engaged in this research in order to generate sitesaturation mutagenesis libraries in difficult templates that could not be randomized with standard methods. the result of our work is that we have produced a generally applicable protocol where we have methodically defined parameters for number of cycles per stage, antiprimer position, and primer concentration, each of which contribute to the successful outcome of the reaction. primer location and orientation as enhancers of amplification reaction are a new concept in optimizing this type of pcr, which provide a new option in cases where standard protocols and optimization methods have failed. we anticipate that the method we present here will be of considerable interest to molecular biologists and protein engineers. protein engineering protocols (methods in molecular biology) directed enzyme evolution: screening and selection methods directed evolution library creation: methods and protocols complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a bacillus subtilis esterase 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efficient one-step sitedirected and site-saturation mutagenesis protocol acknowledgement this research was supported by the german-israeli project cooperation (dip), the deutsche forschungsgemeinschaft (schwerpunkt 1170; "directed evolution to optimize and understand molecular biocatalysis"; project re 359/13-1) and the fonds der chemischen industrie.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-354904-7gq2e6f0 authors: staroverov, sergey a.; volkov, alexei a.; mezhenny, pavel v.; domnitsky, ivan yu.; fomin, alexander s.; kozlov, sergey v.; dykman, lev a.; guliy, olga i. title: prospects for the use of spherical gold nanoparticles in immunization date: 2018-11-06 journal: appl microbiol biotechnol doi: 10.1007/s00253-018-9476-5 sha: doc_id: 354904 cord_uid: 7gq2e6f0 recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. we used spherical gold nanoparticles (average diameter, 15 nm) as a platform for the antigen for swine transmissible gastroenteritis virus (tgev). the literature data demonstrate that immunization of animals with the tgev antigen coupled to gold nanoparticles (gnps) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. the contents of γ-ifn, il-1β, and il-6 in animals immunized with gnp-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. the increased concentration of il-1β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il-6 indicates that the injected preparations are stimulatory to cellular immunity. immunization with the tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals’ immune organs and restores the morphological–functional state of these organs. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. these results can be used to develop vaccines against this infection by employing tgev antigen coupled to gold nanoparticles as a carrier. swine transmissible gastroenteritis is an acute, rapidly spreading, highly contagious enteric disease of pigs of all ages. it is characterized by vomiting, watery and yellow diarrhea, weight loss, and rapid dehydration, leading to a high mortality rate, especially in piglets (mortality decreases with age). swine transmissible gastroenteritis is caused by transmissible gastroenteritis virus (tgev) of the genus coronavirus in the family coronaviridae. the major route of virus transmission is fecal-oral. infected animals cannot digest food properly and die from dehydration. the disease causes great economic losses in pig farms all over the world, and the primary means of animal protection against it is vaccination (cheng and niu 1992; straw et al. 2000) . therefore, the development of new vaccines and the implementation of large-scale vaccination programs are important in pig breeding (moxley and olson (1989) . in several countries, vaccination against swine transmissible gastroenteritis is conducted effectively but only with inactivated monovalent and combined vaccines not recognized internationally. traditional inactivated vaccines have high contents of contaminant substances (up to 99%), causing side effects, and/or may contain, as inactivating compounds, toxic agents such as phenol and formaldehyde, normally used to inactivate microbial cells. moreover, traditional vaccines are usually low immunogenic, because killed microorganisms cannot induce fully functioning cellular immunity. therefore, there is a need to develop new, more effective, and less costly vaccines against swine transmissible gastroenteritis (mout et al. 2012) . recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. in particular, this can be attributed to the development of biocompatible inorganic nanomaterials-gold nanoparticles. gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity. in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. several reports have described the development and use of nanostructures for the delivery of biologically active agents to target cells (mout et al. 2012; bhattacharya and mukherjee 2008; staroverov et al. 2012 ). to reach their target cells in vivo, nanoparticles must bypass many barriers that protect animals against antigens (dreaden et al. 2012) . gold nanoparticles (gnps) have been used widely as carriers of antigens and therapeutic agents in human medicine, veterinary practice, and biology . of particular interest is the ability of gnps to induce humoral immune response to weakly immunogenic antigens and haptens (dykman et al. 2010a; dykman et al. 2010b ). m o r e o v er, g n p s c on j u ga t ed t o t g e v h a v e a n immunostimulatory effect (dykman et al. 2010b) . we studied the immunological variable and morphological changes in the immune organs of guinea pigs immunized with tgev antigen conjugated to gnps as a nanocarrier. antigen tgev strain vn-96 was taken from the museum of strains of the laboratory of virology (scientific research veterinary institute, russian academy of sciences, saratov) and deposited in the all-russian state collection of microbial strains used in veterinary and animal husbandry by the number bvn-96^(patent ru2009124039a 2009). tgev strain vn-96 with a titer of 6.5-7.5 lg tcd 50 ml −1 was used. test cultures were inoculated with the same strain at 4.0 lg tcd 50 ml −1 after 72 h of cultivation. tgev was purified in a sucrose gradient according to horzinek et al. (1982) . the resultant virus suspension was used for protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) at a constant voltage of 80 v for 20 min and then at 200 v for 3 h (maniatis et al. 1982) . for molecular m a s s d e t e r m i n a t i o n s , p r e c i s i o n p l u s p r o t e i n ™ kaleidoscope™ prestained protein standards no. 1610375 were used. the results of protein separation were processed with the gel-imager and gel explorer, version 1.0, programs (dna-technology, moscow). genome identity was checked by polymerase chain reaction (pcr) using a specific primera conservative part of tgev's s genome, containing 886 base pairs (bp) (herrington and mcgee 1993) . rna was isolated from tgev by using a nucleos+tm suspension silicate sorbent (biokom, moscow) according to the manufacturer's instructions. pcr results were recorded on a transilluminator with a dna-analyzer video system. pcr products obtained from a preparation of the tgev strain vn-96 were analyzed by electrophoresis in agarose gel (1.5%) using dna ladder 100 as a molecular size marker and dna of bacteriophage λ as a negative control. as viral nucleic acid is responsible for virulence, it was inactivated, after being isolated from tgev and purified, with ribonuclease (rnaase), a hydrolase enzyme that catalyzes rna hydrolysis. the inactivation was done by the procedure described by fedorova et al. (2011) . the antigen resulting from nucleic acid inactivation was used for animal immunization. spherical gold nanospheres (average diameter, 15 nm) were made by the citrate method of frens (1973) . for reduction, 242.5 ml of 0.001% aqueous tetrachloroauric acid (haucl4; sigma-aldrich) was heated under reflux to 100°c on a magnetic stirrer in an erlenmeyer flask. this was followed by the addition of 7.75 ml of 1% aqueous sodium citrate to the flask. the particle diameter was found as described earlier (khlebtsov and dykman 2010) , by using a libra 120 transmission electron microscope (carl zeiss, germany), a specord s 250 spectrophotometer (analytik jena, germany), and a zetasizer nano-zs particle size and zeta potential analyzer (malvern). to prepare antigen-gold nanoparticle conjugates, we found the bgold number^(minimal amount of antigen that protects the sol against salt aggregation) for the antigen solution. to this end, 20 μl of an antigen solution in pbs (initial concentration, 1 mg ml −1 ) was titrated twofold on a 96-well microtiter plate. each well received 200 μl of gnps and 20 μl of 1.7 m nacl. the minimal stabilizing concentration for the antigen was 12.5 μg ml −1 . conjugation was done by simple mixing (without the use of coupling agents) (dykman et al. 2008 ). thirty six male guinea pigs, weighing 300-350 g at the time the experiment started, were used. the animals were cared for and handled in compliance with the guide for the care and use of laboratory animals, the european convention for the protection of vertebrate animals used for experimental and other scientific purposes, and the legislation of the russian federation. for immunobiological studies, the guinea pigs were divided into four groups of nine each. the animals were immunized subcutaneously along the spinal column by two injections with an interval of 10 days between. the guinea pigs in group 1 were injected with 1.0 ml of tgev antigen-gnps (antigen concentration, 12.5 μg per animal). group 2 received 1 ml of tgev antigen solution (12.5 μg per animal). group 3 served as the control, which received 1 ml of physiological saline. group 4 served as the control gnps, which received 1 ml of gnps solution. gnp-antigen group was in comparison with group which received only physiological saline (control group). the animals were killed 10 days after the last injection, and blood serum samples were taken for immunological studies. immediately after removal, all pieces of tissues and organs were placed in a fixing solution (10% aqueous neutral formalin), and then 15-μm-thick histological sections were prepared on a freezing microtome, model 2515 (reichert wien, austria). all histological samples were stained with hematoxylin-eosin. for isolating peritoneal macrophages, the animals were killed and then fixed on their backs. an incision was made along the midline of the anterior abdominal wall, and the skin flap was carefully separated, with care taken to keep the peritoneum intact. after a puncture had been made with a needle connected to a syringe, 50 ml of pbs, ph 7.2, was injected into the peritoneal cavity. the anterior abdominal wall was then gently massaged, and after 5-7 min, peritoneal fluid was collected with a pasteur pipet through a cut made in the peritoneum and was filtered into a test tube through a nylon filter. the cells were washed three times by centrifugation in pbs at 750 g, after which they were resuspended in 1 ml of pbs and counted in a goryaev chamber. peritoneal macrophages were cultured by standard procedures (leiter 2001) . for isolating splenic lymphocytes, an incision was made along the white line of the peritoneum after peritoneal macrophages had been isolated, and the spleen was removed. the spleen was minced with scissors, and the tissue pieces were mashed through a fine sieve into a petri plate containing sterile pbs. the resulting suspension was subjected to ficoll-urografin density-gradient centrifugation. the lymphocyte ring was collected in a new test tube. the lymphocytes were washed three times by centrifugation in pbs, ph 7.4, at 750 g for 10 min, and the cell sediment was resuspended in 1 ml of pbs. the lymphocytic cells were counted with a haemascreenvet hematology analyzer (hospitex diagnostics, italy). the titer of antibodies in the sera was estimated by enzyme-linked immunosorbent assay (elisa) with horseradish peroxidase-labeled antibodies against guinea pig igg (jackson immunoresearch, uk). the synthetic peptide was used as the immobilized antigen. the reaction results were recorded on a plate screen microplate spectrophotometer. the interleukin concentration in the sera was measured with a plate screen analyzer (hospitex diagnostics, italy) and using reagents of il-1β, il-6, and inf-γ (vector-best, russia). respiratory activity was measured conventionally (bernas and dobrucki 2000) by the ability of cells to reduce nitrotetrazolium blue [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, mtt (sigma-aldrich)] to formazan. briefly, suspensions of known concentrations of isolated animal cells (macrophages and lymphocytes) were centrifuged at 1000 g for 10 min, and the sediment was resuspended in 1 ml of 0.05% mtt and incubated at 37°c for 1 h. after incubation, the cells were centrifuged at 4000 g for 10 min and the sediment was resuspended in 0.5 ml of dimethyl sulfoxide (fluka, switzerland). the amount of reduced formazan was measured with a genesys 10s uv-vis spectrophotometer at 490 nm. to construct the calibration curve, we used commercial formazan (sigma-aldrich) at 0.002, 0.02, 0.2, and 2 mg ml −1 . for studying possible morphological changes in the spleen as an important part of the macrophage system, the animals were immunized with tgev antigen-gnps. the control group comprised of nonimmunized animals. we used the spleens of guinea pigs of the same physiological age. longitudinal and transverse spleen sections (15-μm-thick) were prepared on a freezing microtome, model 2515 (reichert wien, austria), by following standard procedures. histological sections were differentially stained with hematoxylin-eosin. the results were statistically processed by the standard procedures using student's t test to evaluate the reliability of the differences between samplings in the experimental and control studies. after the arithmetic mean and the standard deviation for a given data sample were found, we determined the standard error of the arithmetic mean and its confidence limits by student's t coefficient (n, p) at a significance level of 95% (p = 0.05). the obtained results were statistically processed by the standard procedures integrated in excel 2007 software (microsoft corp., usa). after the arithmetic mean and the standard deviation for a given data sample had been found, we determined the standard error of the arithmetic mean and its confidence limits with account of student's t coefficient (n, p) [number of measurements n = 9, significance level = 95% (p = 0.05)]. these results are presented as histograms. the significance of differences between individual samples was evaluated by a two-sample unpaired student's t test with unequal variances. differences were considered significant when the experimentally found p exp value was ≤ 0.05. the reliability of the changes recorded in the results of each of the experiments described above for the entire set of antigen formulations examined was assessed by one-way analysis of variance (anova) by using fisher's ratio test. the dependences found were considered significant at f > f crit , where the critical value f crit at n = 9 and m = 4-5 (number of data sets) corresponded to p = 0.05 (with the number of degrees of freedom (df) lying between 4 and 14) and the effective value p eff was < 0.05. swine transmissible gastroenteritis is caused by an rna virus belonging to group 1а of the genus alphacoronavirus in the family coronaviridae. the virion has a spherical shape with a diameter of 75-160 nm (martins et al. 2013) . the viral nucleocapsid is a flexible spiral containing single-stranded rna and a large number of nucleocapsid protein molecules (delmas et al. 1986; sturman et al. 1985) . the virus replicates in the cytoplasm of mature epithelial cells located at the ends of the intestine villi. a general scheme of the experiment is depicted in fig. 1 . after the antigen was isolated and its protein composition was analyzed by sds-page, we found that the molecular masses of the separated proteins were identical to those of the tgev proteins. the antigen contained the following proteins: p protein (large surface glycoprotein s; molecular mass, 170-200 kda), n protein (basic nucleocapsid protein, 50-62 kda), and m protein (matrix membrane protein, 25-35 kda) (fig. 2) . a protein fraction of 15-17 kda was also present. according to wesley et al. (1991) , 17 kda is the size of an unglycated structural protein of tgev. thus, all known tgev proteins were separated. the tgev nucleic acid was identified by pcr using a specific primer-a conservative part of tgev's s genome, containing 886 bp. analysis of the pcr products from a purified preparation of the tgev strain vn-96, which was done by agarose gel electrophoresis with dna ladder 100, demonstrated sufficiently high separation of cdna (fig. 3) . the presence of only a characteristic cdna fraction (size, 886 bp) indicated the presence of the tgev genome in the sample. after the virus's nucleic acid was inactivated with ribonuclease, the resultant antigen (a mixture of viral capsid proteins) was used for conjugation with gnps and for subsequent animal immunization. the gnp diameter was measured by tem, the average particle size was 15.7 nm. analysis of the antibody titer data showed that the titer produced by immunization with gnp-antigen conjugates (1:15018.67; p = 0.0014) was higher than that produced by immunization with unconjugated tgev antigen (1:6826.67). in immunology, the mononuclear phagocyte system (mps) (also known previously as the reticuloendothelial or macrophage system) is part of the immune system that consists of phagocytic cells located in reticular connective tissue. the cells are primarily monocytes and macrophages, which accumulate in lymph nodes and in the spleen. the effectiveness of interaction of the gnp-antigen conjugate with cells of the reticuloendothelial system was evaluated using the mtt test. on day 10 after immunization, an increase in the respiratory activity of macrophages was noted (fig. 4) . after immunization with the gnp-antigen conjugate, the activity increased by 97% (p = 0.003) as compared with the control and by 75% (р = 0.0015) as compared with the immunization with tgev antigen and 79% (р = 0.006) as compared with gnp. a study of the respiratory activity of splenic lymphoid cells (fig. 5) showed that after immunization with the conjugate, the activity increased 2.2-fold, as compared to the control, whereas after immunization with tgev antigen alone, it did not change much. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. this indicates that gnps can present antigens to the organs of the endothelial system. the effect of the conjugate on humoral immunity was assessed by comparing changes in the cytokine concentration between the control and experimental animals. there was a statistically significant increase in the content of the γ-ifn in animals immunized with gnp-antigen conjugates (fig. 6) . these animals had the highest content of γ-ifn (59.5 ± 13.6 pgml -1 ; p = 0.023) this group was comparison with group which received only physiological saline (control group). we determined the p value error probability when deviating from the null hypothesis (errors of the first kind). we determined that for our research, p was ≤ 0.05.when the animals were given the native antigen and gnps, no appreciable changes in γ-ifn content were found. the content of il-1β in animals immunized with gnpantigen conjugate was 120.4 ± 23.28 pgml -1 (p = 0.05), which is higher than it was in intact animals (91.04 ± 17.01 pgml -1 ) or in animals given the antigen alone (91.46 ± 17 pgml -1 ; p = 0.96) and gnps (107 ± 20.6 pgml -1 ; p = 0.16) (fig. 7) (p value for gnp-antigen group was comparison with group which received only physiological saline (control group). gnp-antigen group was comparison with group which received only physiological saline (control group). the increased concentration of il-1β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. this point is so important, since it is desirable to avoid an inflammatory process during immunization, which results in the appearance of granulomas. similar data were obtained with il-6 ( fig. 8) . this fact indicates that the injected preparations are stimulatory to cellular immunity. it is known that interferons, which possess protective properties, are produced in the body only when the virus enters or tumor diseases. that is why the analysis of the effect of immunization with the help of gold nanoparticles on the change in the level of interferon was made. it was found that in the immunized group, the concentration of interferon increased by 74% (compared to the control) against 29% for the group immunized with the antigen. when immunizing animals with native antigen, no significant changes in the concentration of interferon in blood serum of the immunized animals were observed. thus, an increase in the concentration of interferon indicates the stimulation of the introduced drugs of the cell link of immunity. the results obtained correlate with research in this area by other scientists. for example, in work (zhang et al. 2018) showed that tgev n protein induced er stress and activated nf-κb, which was responsible for the upregulation of il-8 markers protein markers (kda) protein p protein and bcl-2 expression. tao et al. (2017) also revealed an increase in the concentration of cytokines including interferon gamma and interleukin 1 in response to immunization aunp-m2e + scpg. but he mainly considered the reaction of splenocytes to a given complex (their reaction, which was expressed in the production of cytokines, on the effect of aunp) was studied. in addition, aunp-m2e was administered together with scpg which is a strong immunomodulator. it was interesting for us to look at the production of these cytokines in the body as a whole and to reveal the general clinical changes that can develop on the introduction of antigen and aunp. one can speculate that gnps facilitate antigen expression with the major histocompability complex on the surface of antigen-presenting cells. this subsequence ensures that the viral peptides are effectively presented to cytotoxic t lymphocytes and natural killers. our data for the increase in γ-ifn activity are in agreement with those for the increase in the respiratory activity of splenocytes on day 10 after the last injection, another indication that colloidal gold can stimulate the release of γ-ifn by t cells, thereby enhancing the activity of lymphoid immunity. in guinea pigs, the spleen forms leucocytes and removes old red blood cells (erythrocytes). the spleen framework is formed by trabeculae, which consist of reticular connective tissue, smooth muscle cells, and blood vessels (at the same time, the spleen contains only efferent lymphatic vessels). the reticular connective tissue includes both red and white pulp. the white pulp is a lymphoid tissue that produces immune and blood cells. the red pulp functions to filter blood for antigens, microorganisms, and defective red blood cells; therefore, it contains various blood corpuscles, including large and small lymphocytes, immature leucocytes, leucocytes at the final stage of graininess, and disintegrating erythrocytes. the spleen is also important for the development of humoral immunity and is a b-system organ. in the animals immunized with gnp-antigen conjugates, the lymphoid follicles had distinct boundaries and contained large number of densely spaced lymphoid cells (fig. 9c . the lymphoid tissue was well structured and was located in a compact manner. there was a clear boundary between the red and white pulp regions, and there were full of cellular elements. for comparison, we analyzed morphological changes in the spleens of the animals immunized with tgev antigen alone. the observed changes were characterized by the existence of perivascular edema (fig. 9d) , sometimes accompanied by proliferation and desquamation of the vessels' endothelium. moreover, the lymphoid tissue of the follicles became sparse, and the number and density of lymphoid cells decreased. in spleen tissue sections from the animals immunized with tgev antigen alone, the boundary between red and white pulp was less clear, the size and total number of follicles in the microscopic field decreased, and no germinative centers could be detected. these results point to degradative processes in the immune organs of the animals. in the control group of guinea pigs and gnps, injected with physiological saline, no morphological changes in spleen tissue were observed (fig. 9a, b) . gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity (pissuwan et al. (2008) . in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. this quality allows the use of gold nanoparticles for a variety of purposes, including vaccine delivery (versiani et al. 2016 ). there are a series of works devoted to the use of gold nanoparticles as carriers of antigens for pathogens such as the virus grippe (tao et al. 2014) , virus foot-and-mouth (chen et al. 2010) , tetanus toxoid (barhate et al. 2013) , and yersinia pestis (gregory et al. 2012) . in our work, we used spherical gold nanoparticles (average diameter, 15 nm) as a platform for the antigen for tgev. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid a b c d fig. 9 a spleen tissue section from the animals immunized gnps. hematoxylin-eosin staining; magnification, × 50. b spleen tissue section from the animals control group. hematoxylineosin staining; magnification, × 50. c spleen tissue section from the animals immunized with tgev antigen-gnps. hematoxylin-eosin staining; magnification, × 50. lymphoid follicles have distinct boundaries. d spleen tissue section from the animals immunized with tgev antigen alone. hematoxylineosin staining; magnification, × 50. perivascular edema and sparse lymphoid tissue in the follicles can be observed fig. 8 concentration of il-6 in animals immunized with gnpantigen conjugates (tgev antigen р = 0.95; gnp-antigen conjugate р = 0.002; gnps р = 0.08) (antibody-forming) cells. the contents of γ-ifn, il-1β, and il-6 in animals immunized with gnp-antigen conjugates were higher than those in the intact animals or in animals given the antigen alone. the increased concentration of il-1β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il-6 indicates that the injected preparations are stimulatory to cellular immunity. several reports have described the development and use of nanostructures for stimulating cellular immunity. for example, xu et al. (2012) showed that gold nanorods associated with the dna vaccine may cause increased formation of γ-ifn in hiv-infected mice. assis et al. (2018) showed that gold nanorods, functionalized with cysteamine and associated with the protein rsm29, are stimulatory to il-1. in a work, tao et al. (2017) showed that the use of nanostructured vaccine led to an increase in the concentration of igg2a, iga, and il-1a, il-2, il-17, tnf-и rantes. our research correlates with these studies but in the present work we used gold nanoparticles for the first time as a platform for antigen for tgev. also, we did nonspecific adsorption of the antigen and did not perform a chemical surface functionalization. this made it possible to simplify the conjugation process, as well as to remove additional procedures related to the purification of the drug from toxic components. additionally we analyzed the morphological changes in the spleens of the animals immunized with gnp-antigen conjugates and tgev antigen. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, 2000) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. in conclusion, immunization with tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. the results of this study can be used in the further development of nanovaccines against swine transmissible gastroenteritis. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, 2000) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. funding information this study was funded by the russian science foundation (project no. 18-14-00016). conflict of interest the authors declare that they have no conflict of interest. ethical statement all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. the use of gold nanorods as a new vaccine platform against schistosomiasis quillajasaponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies the role of plasma membrane in bioreduction of two tetrazolium salts, mtt, and ctc biological properties of bnakedm etal nanoparticles assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide investigation on the porcine epidemic diarrhea prevalent on qinhai antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer 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nanorods: promising dna vaccine adjuvant for hiv-1 treatment transmissible gastroenteritis virus n protein causes endoplasmic reticulum stress, upregulates interleukin-8 expression and its subcellular localization in the porcine intestinal epithelial cell key: cord-011212-ovjdzyxv authors: pan, qing; wang, jing; gao, yulong; cui, hongyu; liu, changjun; qi, xiaole; zhang, yanping; wang, yongqiang; li, kai; gao, li; wang, xiaomei title: development and application of a novel elisa for detecting antibodies against group i fowl adenoviruses date: 2019-12-14 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-10208-3 sha: doc_id: 11212 cord_uid: ovjdzyxv since 2015, outbreaks of hepatitis-hydropericardium syndrome (hps) caused by a novel genotype of fowl adenovirus 4 (fadv-4) infection have created serious economic losses in china. given that other serotypes of hypervirulent fadvs have also been reported in poultry around the world, a common elisa for all serotypes within the group i fowl adenoviruses (fadv-i) is urgently needed, especially for clinical epidemic serotypes. in this study, we used high purity and concentration virions of fadv-4 and developed a common elisa for detecting antibodies against 12 fadv-i serotypes. the developed elisa was able to distinguish between antibodies against fadv-i, fadv-iii, and other heterologous viruses without any cross-reaction. furthermore, the elisa showed higher sensitivity than the fadv-1-based elisa to the novel fadv-4 found in china. moreover, since there are no commercial vaccines against fadvs in china, the elisa was applied to detect sera samples from specific pathogen-free chickens inoculated with inactivated fadv-1, fadv-4, and fadv-8a. the assay showed high sensitivities for all three detected serotypes within fadv-i. in conclusion, a novel, common elisa for fadv-i was developed in this study and could be a powerful tool for seroepidemiological investigations and fadvs vaccine development. fowl adenoviruses are divided into three groups, group i fowl adenovirus (fadv-i) contains five species (a to e) with 12 serotypes (1-8a, 8b-11) (hess 2000) isolated from fowls, group ii (fadv-ii) includes the hemorrhagic enteritis virus (hev) of turkeys and the marble spleen disease virus (msdv) of pheasants (domermuth et al. 1980) , and group iii (fadv-iii) is mainly associated with egg drop syndrome virus (edsv) (huang et al. 2015) . inclusion body hepatitis (ibh), hydropericardium syndrome (hps), and gizzard erosion (ge) associated with fadv-i infection have been reported worldwide, e.g., in pakistan (khawaja et al. 1988; mansoor et al. 2009 ), chile (toro et al. 1999) , korea (choi et al. 2012) , canada (dar et al. 2012) , hungary (kaján et al. 2013) , india (mittal et al. 2014) , japan (mase and nakamura 2014) , south africa (joubert et al. 2014) , mexico (vera-hernández et al. 2016) , poland (niczyporuk 2016) , and china , resulting in significant economic losses to the poultry industry. hps is caused by fadv-4 (pan et al. 2017a ), whereas ibh is always related to fadv-2, fadv-11, fadv-8a, or fadv-8b (morshed et al. 2017) and ge is mainly caused by fadv-1 (matczuk et al. 2017) . notably, severe hps, with a high mortality rate of 20-80%, caused by a novel genotype fadv-4 has been widespread in china since 2015 (li et al. 2016; pan et al. 2017a ) and is a major threat to the poultry industry. in recent years, a variety of fadvs detection methods have been developed for large-scale seroepidemiological investigations and hps vaccine evaluation. for antigen detection, polymerase chain reaction (pcr) (günes et al. 2012) , quantitative pcr (qpcr) (pan et al. 2017c ), high-resolution melting (hrm) curve analysis (steer et al. 2009 ), loop-mediated qing pan and jing wang contributed equally to this work. * xiaomei wang wangxiaomei@caas.cn isothermal amplification zhai et al. 2019) , and sandwich enzyme-linked immunosorbent assays (elisa) (shao et al. 2019a; shao et al. 2019b ) have been developed. the traditional methods for fadvs serological diagnosis are agar gel precipitation (agpt) and virus neutralization (vn) tests. while agpt is generally less sensitive, the process of vn is not conducive to rapid and large-scale diagnosis. thus, a simple, rapid, and sensitive diagnostic method is urgently required for fadvs detection, for which elisa, which is widely used in large-scale serological investigation, is simple to operate, and has high sensitivity, is an excellent candidate technology. prevention and control of hps have been attempted, mainly through the use of inactivated virus pan et al. 2017b) or subunit vaccines schachner et al. 2014; shah et al. 2017; wang et al. 2018) , for some fadv serotypes. the immune response to vaccines is generally monitored by the presence of an antigen-specific antibody and the neutralization antibody. in one recent study, recombinant fiber-based indirect elisa was used to detect serum samples from chickens experimentally inoculated with different fadv-1 or fadv-4 strains (feichtner et al. 2018) . a recombinant hexon-based single serum dilution elisa was also developed to measure the hexon-specific antibodies against fadv-4 in sera of chickens (rajasekhar and roy 2014) . both of the elisas mentioned above were fadv serotypespecific and based on variable recombinant proteins. however, some other fadv serotypes, such as fadv-1 and fadv-8a, have also been shown to cause serious economic losses to the poultry industry. unfortunately, there is currently no commercial fadv-i elisa kit in china, thus a common elisa for all fadv-i serotypes is urgently needed. in this study, we developed a group-specific and sensitive elisa based on the novel genotype of fadv-4 for detecting antibodies against twelve fadv-i serotypes. the common elisa provides a powerful tool for the seroepidemiological investigations and vaccine development. as we reported previously (pan et al. 2018) , the hljfad15 strain (genbank no. ku991797) was isolated from layers and identified as serotype 4 (fadv-4). the fadv-1 celo (genbank no. u46933) and fadv-8a (a clinical isolate) strains were deposited at the state key laboratory of veterinary biotechnology, harbin veterinary research institute (harbin, china). chicken leghorn male hepatocellular (lmh) cells were kindly gifted by prof. guozhong zhang (china agricultural university, beijing, china) and were cultured in dulbecco's modified eagle's medium (dmem) (thermo fisher scientific, waltham, ma, usa) supplemented with 10% fetal bovine serum (fbs) (gibco, san diego, ca, usa), 100 iu/ml penicillin, and 100 μg/ml streptomycin. following hljfad15 (fadv-4) strain propagation and purification, the fbs concentration in confluent cultures was reduced to 2% for maintenance. the lmh cells were infected with 0.1 multiplicity of infection (moi) of fadv-4 and incubated at 37°c in a 5% co 2 atmosphere for 3 days. formaldehyde was added to the culture medium for virus inactivation, and viral particles were harvested from virusinfected lmh cells at a concentration of 0.2% in the final product (kim et al. 2014) . culture fluids were collected and, after 3 cycles of freezing and thawing, the mixture was centrifuged at 5000×g for 15 min to remove cellular debris. supernatants were transferred to 20%, 30% (w/w) sucrose solution and centrifuged at 30,000 rpm for 3 h using a beckman sw 32 rotor in a model optima xpn-100 ultracentrifuge (beckman coulter, brea, ca, usa). virus pellets were collected and suspended in phosphate-buffered saline (pbs). culture suspensions were collected, purified in 1.33 g/ml cesium chloride (cscl 2 ) (amresco, solon, usa), and centrifuged at 35,000 rpm for 16 h using a beckman sw 55 rotor. two discrete bands were formed following final ultracentrifugation. the bands were aspirated with a syringe by puncturing the side of the tube, suspended in pbs, centrifuged at 32,000 rpm for 2 h using a beckman sw 32 rotor, and culture fluids were collected (pan et al. 2018) . the morphology of fadv-4 preparations was verified by electron microscopy. carbonate buffer (ph = 9.6), tris-hcl buffer (ph = 8.0), and phosphate buffer (ph = 7.4) were used as coating buffers. pbst containing 5% skim milk, pbs containing 5% bovine serum, and pbs containing 5% gelatin were used as blocking buffers. fadv-4 stocks with a concentration of 1.03 mg/ml, as measured by micro-volume spectrophotometer (implen, munchen, germany), were obtained and prepared into working dilutions (1 μg/ml, 3 μg/ml, and 5 μg/ml) using coating buffer. the working dilutions were added into microtitre plates (100 μl/well) and incubated at 4°c for 12, 16, or 20 h. after incubation with the coating antigen, the plates were washed three times with pbs containing 0.05% tween-20 (pbst) and then incubated with blocking solution at 37°c for 1, 2, or 3 h. after three washes, serum samples were diluted 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1,600, and 1:3200 and incubated at 37°c for 0.5, 1, and 2 h, respectively. following incubation, samples were washed three times and incubated at 37°c for 0.5, 1, and 2 h with hrp-conjugated rabbit anti-mouse antibodies (sigma, missouri, usa) diluted 1:2500, 1:5000, and 1:10,000, respectively. after washing, 100 μl tetramethylbenzidine (tmb) substrate (amresco, solon, usa) was added to each well and the plates were incubated in the dark for 5, 8, and 11 min. the enzymatic reaction was quenched by hydrofluoric acid and the optical density (od) was determined at 630 nm. ods presented represent the mean from duplicate wells. the optimal conditions were determined by evaluating the od values and the positive/negative ratio (p/n) of the samples. the cut-off was determined according to the sample/positive (s/p) ratio by calculating the arithmetic mean plus three times the standard deviation (sd). forty specific antibody-negative chickens were divided into four groups: fadv-1 immunization group (n = 10), fadv-4 immunization group (n = 10), fadv-8a immunization group (n = 10), and a control group of age matched unvaccinated chickens (n = 10). the formaldehyde inactivated antigen solution was emulsified with oil adjuvant at a ratio of 25:75 (w/w). the immunization group was immunized intramuscularly with 0.5 ml inactivated viruses containing 10 6 tcid 50 fadv-1, fadv-4, fadv-8a antigens per chicken at the age of 21 days. five serum samples of chickens in each group were collected weekly post inoculation until the termination of the experiment. in total, 850 clinical serum samples were examined by the developed elisa for the presence of fadv antibodies. these samples were collected from breeder flocks of unknown disease and vaccination status in jiangsu, heilongjiang province. fadv antibodies were detected by the common elisa and results were compared with a commercial kit (biochek, reeuwijk, the netherlands) for fadv-i. anti-fadvs antibodies were detected by indirect immunofluorescence assay (ifa). an ifa was carried out on lmh cells plated in 24 well plates and infected with 0.1 moi of the fadv-4. after 2-day incubation at 37°c in a 5% co 2 atmosphere, the wells were fixed with iced ethanol for 20 min at room temperature and washed three times with pbs. clinical serum samples diluted 1:10 in pbs were used as primary antibodies for 1 h at 37°c. after washing three times, alexa fluor tm 488 goat anti-mouse igg (h+l) secondary antibody (1:200) (invivogen, san diego, california, usa) was added, followed by incubation for 1 h at 37°c. after washing three times, the wells were investigated by evos f1 inverted fluorescence microscope. differences between the two groups were evaluated by student's t test and considered significant at ** p < 0.01. purified intact virus particles were used as an optimal coating antigen to develop a common elisa in this study. highpurity virions were collected by sucrose and cscl 2 density gradient centrifugation. subsequently, fadv-4 stocks with a concentration of 1.03 mg/ml, as measured by micro-volume spectrophotometer, were obtained. the fadv-4 virions were verified by electron microscopy, with negatively stained preparations showing nearly round surface with a diameter of 70-90 nm. as shown in fig.1 , the purified virions were intact and of high purity, and the preparation had good dispersion and was evenly distributed throughout the field. fig. 1 the morphology of the purified virus particles from cell-cultured hljfad15 (genbank no. ku991797) by electron microscopy the optimum working range of antigen and serum were decided by checkerboard titration and determined by the greatest p/n ratio. antigen concentrations of 3 μg/ml were added to microtitre plates coated with 100 μl in carbonate buffer (ph 9.6). following incubation for 16 h at 4°c, the plates were washed three times with pbst and blocked with pbst containing 5% non-fat milk for 2 h at 37°c. serum samples diluted 1:100 in pbs were incubated for 30 min at 37°c. after washing three times, secondary antibodies were incubated for 30 min at 37°c before a final washing step. subsequently, 100 μl tmb substrate was added to each well and the plates were incubated in the dark for 5 min. the enzymatic reaction was quenched with 100 μl 0.5 m hydrofluoric acid and the od values were determined at 630 nm. based on the arithmetic mean s/ p value of the 200 clinical negative samples (0.047) and the sd value (0.051), the cut-off for the elisa was determined to be 0.199. serum samples with s/p value greater or less than 0.199 were determined to be positive or negative, respectively. in the specificity assay, the negative control serum collected from non-treated spf chickens was confirmed to be negative by the common elisa developed in this study, and the positive control serum obtained from spf chickens inoculated with hljfad15 was detected to be positive. as shown in fig. 2 , the s/p values of other heterologous viruses, including h5 and h9 aiv, ndv, ibv, and iltv positive sera, were below the established cut-off value (ranging from 0.056 to 0.106) and were hence determined to be negative by the developed elisa. in group-specificity and serotype-specificity assays, edsv (fadv-iii)-positive serum tested by the common elisa was negative (fig. 3a) . different serotypes of fadv-i including fadv-1, fadv-4 gy, fadv-8a, fadv-9, fadv-10, and fadv-11 positive serums ranged from 0.456 to 1.185 (fig. 3b) , above the elisa cut-off value, were determined to be positive. sensitivity analyses comparing the fadv-4 elisa and fadv-1 elisa using serum samples collected from chickens infected with hps showed a significantly higher sensitivity of the elisa developed based on fadv-4 in this study than the fadv-1-based elisa. as shown in fig. 4 , the limit of detection (lod) of the common elisa developed for hljfad15 (fadv-4) positive sera was 1:25,600 while the elisa based on fadv-1 was 1:6400. the common elisa showed higher s/p values than the fadv-1-based elisa detecting antibodies against fadv-4 positive sera with each dilution assayed. kinetic analyses of specific antibody responses following inoculation of all three fadv hypervirulent serotypes were assessed by the common elisa. fadv-i antibodies in the fig. 2 fadv-specificity assay for the common elisa using different heterologous viruses, including h5 and h9 aiv, ndv, ibv, iltv. spf chicken serum as negative control; fadv-4 positive serum as positive control fig. 3 group-specificity and serotype-specificity assay for the common elisa using edsv (fadv-iii) (a) and different serotypes of fadv-i, including fadv-1, fadv-4 gy, fadv-10, fadv-9, fadv-11, and fadv-8a, respectively (b). spf chicken serum as negative control; fadv-4 positive serum as positive control negative control group were all negative when tested by the common elisa. as shown in fig. 5 , serum antibody titres of the experimentally inoculated chickens increased with the time of immunization. in chickens inoculated with inactivated fadv-1 (fig. 5a) , fadv-4 (fig. 5b) , or fadv-8a (fig. 5c) , the earliest detection of antibodies occurred 2 weeks after inoculation (p < 0.01). two weeks after immunization with inactivated virus, the positive rate of antibodies against all three detected serotypes within fadv-i reached 100%. the antibodies against the emergent novel fadv-4 showed higher s/p values than antibodies against fadv-1 and fadv-8a. in field sera, the common elisa detected 69.1% (587/850) positives, whereas the commercial fadv-i elisa kit test detected 72.2% (614/850) positives (table 1 ). the positive coincidence rate between the commercial kit and the developed elisa was 92.99% and the negative coincidence was 89.92%; the total coincidence was 92.43%. anti-fadvs antibodies were detected by ifa. the common elisa and ifa detected 71.3% (107/150) and 71.3% (107/150) positives, respectively, and the total coincidence between the common elisa and ifa was 100% (data not shown). hps induced by fadv-4 has emerged across several different areas in china since 2015 (niu et al. 2018; zhao et al. 2015) and caused serious economic losses and poses a great threat to the poultry industry. the pathogenic fadv-4 strain has been isolated and characterized as a novel genotype (ye et al. 2016) . however, there are currently no commercial fadv-i or fadv-4 elisa kits in china for detection of antibodies against the pathogenic fadvs or fadv-4 specifically. many studies have been conducted on fadvs antibody detection using elisa based on fadv-1. in one study, celo (fadv-1) virus was used as a coating antigen to establish an elisa method for detecting fadv-1 and avian adenovirus-associated virus (dawson et al. 1980) . as reported, fadv-4 showed higher cross-reactivity among 10 serotypes than did fadv-1 (calnek et al. 1982) . given that homologous elisa reactions were stronger than heterologous reactions in many cases, we evaluated fadv-4 as a coating antigen to develop a common elisa for detecting all twelve fadv-i serotypes. the elisa developed based on fadv-4 in this study showed considerable cross-reaction within the twelve fadv-i serotypes and higher sensitivity than the fadv-1-based elisa for detecting antibodies against the novel emergent fadv-4. ultimately, we successfully established a common elisa for specifically detecting antibodies against all twelve fadv-i serotypes, which could be a powerful tool for seroepidemiological investigation of ibh, ge, and, especially, the emergent hps in china. unfortunately, there is no commercial vaccine against fadvs, including the fadv-4 strain, and a common detection method is urgently needed to facilitate vaccine development and evaluation. though several recombinant protein-based elisas have been used to detect antibodies in chickens inoculated with subunit vaccines, such as rfiber-1, rfiber-2, and rhexon protein, these elisa were able to detect antibodies against fadv-4-specific (feichtner et al. 2018; he et al. 2018; rai et al. 2005; rajasekhar and roy 2014) . furthermore, prokaryotic expressed proteins need to be renatured to restore tertiary and quaternary spatial structures, which may lead to false-negative results from assays using recombinant proteins. considering that fadv-4 and some other serotypes within fadv-i are pathogenic to chickens worldwide (grgić et al. 2011; lim et al. 2011; morshed et al. 2017 ), a common elisa should be more suitable for fadvs vaccine development. the common elisa developed in our study was applied to detect serum samples from spf chickens inoculated with inactivated fadv-1, fadv-4, and fadv-8a, and showed high sensitivity for all three fadv hypervirulent serotypes. these results suggest that the common elisa developed in our study is capable of monitoring antibodies against all serotypes in variable fadv-i vaccine development and evaluation. in conclusion, the common elisa developed in this study showed considerable cross-reactivity among all 12 fadv serotypes and was capable of detecting specific antibodies against fadv-i. furthermore, the elisa showed higher sensitivity in detecting serum samples of hps caused by the novel fadv-4 genotype that has recently emerged in china. our elisa could be a powerful tool for seroepidemiological investigations and development of vaccines for different fadv-i serotypes. serological crossreactivity of avian adenovirus serotypes in an enzyme-linked immunosorbent assay epidemiological investigation of outbreaks of fowl adenovirus infection in commercial chickens in korea pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in saskatchewan chickens pronovost ad (1980) an enzyme-linked immunosorbent assay for detection of antibodies to avian adenovirus and avian adenovirus-associated virus in chickens incidence and distribution of "avian adenovirus group ii splenomegaly of chickens development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of 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observed in a highly virulent fowl aviadenovirus serotype 4 immune protection efficacy of fadv-4 surface proteins fiber-1, fiber-2, hexon and penton base outbreaks of serotype 4 fowl adenovirus with novel genotype lamp real-time turbidity detection for fowl adenovirus a loop-mediated isothermal amplification coupling with a lateral flow dipstick for rapid and specific detection of fowl adenovirus serotype-4 pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in china key: cord-010914-08omkszy authors: morinaga, kana; yoshida, keitaro; takahashi, kohei; nomura, nobuhiko; toyofuku, masanori title: peculiarities of biofilm formation by paracoccus denitrificans date: 2020-01-30 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10400-w sha: doc_id: 10914 cord_uid: 08omkszy most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. paracoccus denitrificans is a non-motile, gram-negative bacterium that forms a very thin, unique biofilm. a key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein a (bapa), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-gmp or c-di-gmp). cyclic-di-gmp is a major second messenger involved in biofilm formation in many bacteria. though cyclic-di-gmp is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in p. denitrificans. furthermore, quorum sensing (qs) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. the qs signal used in p. denitrificans is hydrophobic and is delivered through membrane vesicles. studies on qs show that p. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. in this review, we discuss the peculiarities of biofilm formation by p. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions. most bacteria in the environment form bacterial communities embedded in extracellular polymeric substance matrix known as biofilms (chan et al. 2016) . biofilms are related to human health as well as infections and have a potential role in colonization of the gut by commensal bacteria which are still largely unexplored (donlan 2001; sekirov et al. 2010) . biofilms are also actively used in applications such as biocatalysts (halan et al. 2012; rosche et al. 2009 ), and in wastewater treatment, where bacteria are present in aggregated forms of cells, also known as activated sludges and granules. the cells inside biofilms show a different physiology from that of the planktonic counterparts, leading to increased resistance to environmental stresses such as ph shift, osmotic shock, and uv radiation (davey and o'toole 2000; flemming 1993) . hence, studying biofilm would lead to our better understanding of the bacterial ecology and optimization of biofilm applications. recent studies highlight the importance of flagella and pili driven motility in biofilm formation. biofilm formation is first initiated by attachment of planktonic cells to the surface; this step is called initial attachment (stoodley et al. 2002) . in many bacteria, motility accelerates surface attachment of cells. initial attachment is followed by irreversible attachment, where cells initiate formation of highly structured microcolonies and embed themselves in eps. in vibrio cholerae, type iv pili, together with flagella, are utilized for solid surface motility and to accelerate surface attachment (utada et al. 2014) , whereas in pseudomonas aeruginosa, type iv pili are required for microcolony formation (o'toole and kolter 1998) . eventually, the subpopulations of cells in the biofilm disperse through motility and leave the biofilm . while flagella and pili driven motility has been shown to play an important role in each step of biofilm formation, little is known about how non-motile bacteria form biofilms, especially gram-negative bacteria. p a r a c o c c u s s p e c i e s a r e g r a m -n e g a t i v e alphaproteobacteria often present in soil, activated sludges, and aerobic granular sludges (cydzik-kwiatkowska 2015; pelissari et al. 2017; xia et al. 2018) . one of the most studied paracoccus species is paracoccus denitrificans that is a nonmotile, denitrifying bacterium and is used as a model organism for studies of denitrification, respiration chains, and polyhydroxyalkanoate production (kelly et al. 2006; kojima et al. 2004; lycus et al. 2018) . in contrast to what has been shown in most bacteria that form highly structured architectures, p. denitrificans forms a peculiarly thin biofilm that consists of almost a monolayer of cells (yoshida et al. 2017) . recent studies show a critical role of an adhesion protein bapa in initiating biofilm formation in p. denitrificans (yoshida et al. 2017) . further studies showed that the biofilm structure is regulated by an intracellular second messenger cyclic diguanosine monophosphate (cyclic-di-gmp) and by bacterial communication (kumar and spiro 2017; morinaga et al. 2018; toyofuku et al. 2017) (fig. 1) . furthermore, p. denitrificans produces a hydrophobic signal that is carried by membrane vesicles (mvs) ). here, we review current knowledge on biofilm formation by p. denitrificans and discuss its associated factors and functions. paracoccus species are shown to form biofilms that have important applications, especially in bioreactors, while the detailed mechanism of biofilm formation in this genus is largely unknown (nisha et al. 2015; singh et al. 2015) . due to their versatile metabolisms, paracoccus species are considered as important organisms for bioremediation and are suggested to be involved in degrading and removing refractory pollutants such as 4-chlorophenol from wastewater (gomez-acata et al. 2018; zhao et al. 2015) . several paracoccus species can carry out denitrification, and are frequently isolated from denitrifying reactor (neef et al. 1996; shi et al. 2013 ). in addition, p. denitrificans is isolated from microbial fuel cell anode biofilm communities and can generate electricity using formic acid as an electron donor (kiely et al. 2010) . furthermore, p. denitrificans can be immobilized in gels with nitrosomonas europaea for efficient nitrogen removal from wastewater and have great potentials in their applications (kokufuta et al. 1988; uemoto and saiki 1996) . hence, understanding biofilm formation in paracoccus species may optimize the functions of biofilm-based bioreactors. biofilm structure of p. denitrificans biofilms are generally a thick layer of cells where microcolonies with mushroom-like structures or mound-like structures could often be observed inside. the thick layer and metabolic activity of cells form microenvironments in biofilms, with micro-scale gradients or patches of chemicals, such as electron acceptors and donors, nutrients, ph, and cell-to-cell communication signals (flemming et al. 2016) . the biofilm architecture could be highly organized, even including water channels to facilitate the transport fig. 1 schematic image of biofilm formation in p. denitrificans. bapa protein is necessary for the attachment to initiate biofilm formation (kumar and spiro 2017; yoshida et al. 2017) . cyclic-di gmp inhibits bapa gene expression or bapa protein secretion (kumar and spiro 2017) . qs inhibits biofilm formation (morinaga et al. 2018) . confocal laser scanning microscopy images show biofilm structure of wildtype p. denitrificans and the pdni mutant. reproduced with permission from (morinaga et al. 2018 ) with modifications of liquids (wilking et al. 2013) . one of the remarkable features of p. denitrificans biofilm is that it forms a very thin and flat biofilm with less than ∼4 μm thickness in a liquid-air interface (yoshida et al. 2017) , making it an interesting model for biofilm formation as compared to other model organisms such as p. aeruginosa that form multilayered biofilms (heydorn et al. 2002; sauer et al. 2002) confocal laser scanning microscopy (clsm) analysis showed that the cells in the biofilm are often densely packed and frequently arranged in chains (yoshida et al. 2017 ). since p. denitrificans is non-motile, it is suggested that the cells spread on the surface by cell division, resulting in dense biofilms with chains of cells. similarly, a monolayer biofilm is also observed in another paracoccus species which biofilm morphology changes depending on the culture conditions (srinandan et al. 2010 ). bapa protein is critical in biofilm formation by p. denitrificans given the thin layer of cells and lack of motility, initial attachment would have more direct impact on the biofilm structure in p. denitrificans compared to other bacteria that undergo several stages to finally form a mature biofilm. bacteria secrete various types of eps to adhere to surfaces and form biofilms. the main eps components are polysaccharides, proteins, lectins, and nucleic acids (flemming and wingender 2010; schooling and beveridge 2006) . bacteria often produce several types of eps that interact with each other and play different roles in forming biofilms. for example, v. cholerae produces an extracellular vibrio polysaccharide (vps), biofilm-associated protein 1 (bap1), and the matrix proteins, rugosity and biofilm structure modulator a (rbma) and rugosity and biofilm structure modulator c (rbmc) (berk et al. 2012; yan et al. 2016) . vps in the biofilms maintain the 3-dimensional biofilm structure by accumulation between cells, whereas bap1 acts as cell-to-surface adhesion and rbma functions as cell-to-cell linkage. bap is a group of proteins that are monomeric repetitive adhesins. baps in gram-negative bacteria are repeats-in-toxin (rtx) family of proteins and composed of three domains: n-termini, core large repeats and c-termini (satchell 2011) . molecular sizes of baps vary, ranging from 1873 to 8682 amino acids (aa), depending on the core repeat sequences (lasa and penades 2006) . baps are localized at cell surfaces and mediate cell-to-surface and/or cellto-cell adhesion. in gram-negative bacteria, baps are secreted directly from the cytoplasm to extracellular space by their abc transporters known as type i secretion system. rtx adhesins contain glycine-aspartate-rich peptide repeats at the c-termini and each peptide binds to a calcium ion (satchell 2011) , which is important at least for some baps to function (martinez-gil et al. 2012) . one of the best studied bap proteins in gramnegative bacteria is lapa, which is involved in cell adhesion of pseudomonas fluorescens (hinsa et al. 2003 ). an analysis of eps components in p. denitrificans using epsdegrading enzymes suggested that an extracellular proteinaceous component influences biofilm formation (yoshida et al. 2017) . in line with this observation, the biofilm formation of p. denitrificans requires bapa for its initial cell attachment (kumar and spiro 2017; yoshida et al. 2017) . bapa protein of p. denitrificans contains a large repeat region consisting of 891 tandem repeats of aspartate and alanine although the precise roles of this extremely acidic sequence are still unknown. this repeat sequence is unique to this protein, while other baps typically contain 80 to 300 aa repeat unit sequences (satchell 2011) . bapa protein of p. denitrificans is secreted extracellularly by type i secretion system bapbcd. bapa is localized at the cell surface and makes the cell surface more hydrophobic, which presumably drives cells to attach to the substratum (yoshida et al. 2017) . it was further shown that bapa is a calcium-binding protein and it is necessary for cell attachment (kumar and spiro 2017) . consistent with these results, divalent cations are shown to enhance biofilm formation in a paracoccus species and edta treatment can detach biofilm formation (srinandan et al. 2010 ). bapa protein regulation in p. denitrificans is not well studied, but nitric oxide (no) has been reported to control the bapa level that partially involves cyclic diguanosine monophosphate (cyclic-di-gmp). cyclic-di-gmp is an intracellular second messenger involved in biofilm formation of many bacteria by controlling eps production and motility. for example, in p. aeruginosa, accumulation of cyclic-di-gmp downregulates cell motility and upregulates eps production (valentini and filloux 2016) . low concentration of cyclic-di gmp due to degradation by phosphodiesterases (pdes) induces cell dispersal from biofilms. the intracellular cyclic di-gmp level is controlled by its synthesis by diguanylate cyclase (dgc), and degradation by pde. cyclic-di-gmp is synthesized from gtp by dgc which has t h e g g d e f d o m a i n s a n d d e g r a d e d t o 5 ′phosphoguanylyl-(3′-5′)-guanosine (pgpg) and/or gmp by pdes which have eal or hd-gyp domains (valentini and filloux 2016) . p. denitrificans possesses two proteins (pden_3720; dgca and pden_3982; dgcb) with ggdef domains and two proteins (pden_0876; pdea and pden_2025; pdeb) with eal domains (kumar and spiro 2017) . dgcb has an n-terminal response regulator domain, implying a signal cascade from a sensor partner. dgca overlaps with a gene coding a protein that has heme-nitric oxide/oxygen binding (h-nox) domain. h-nox protein commonly functions as a sensor for the gaseous signaling agent nitric oxide (no) (plate and marletta 2013) , suggesting that h-nox protein in p. denitrificans regulates the biosynthesis of cyclic-di gmp. p. denitrificans produces no as an intermediate of denitrification. kumar and spiro showed that no stimulates biofilm formation in p. denitrificans (kumar and spiro 2017) . using a series of mutants, they showed that altering the no and cyclic-di gmp levels controls the abundance of bapa protein on the cell envelope and biofilm formation. it is suggested that no controls biofilm formation via both h-nox/cyclic-di-gmp dependent and independent pathways. in other bacteria, no often leads to biofilm dispersal (arora et al. 2015; barraud et al. 2009 ), and the opposite reaction or tolerance against no could be one of the reasons why paracoccus species become abundant in denitrifying reactors (singh et al. 2015) . quorum-sensing down-regulates biofilm formation in p. denitrificans another major regulatory system in biofilm formation of p. denitrificans is quorum sensing (qs). bacteria can communicate with each other using signaling molecules, and the density-dependent bacterial communication is called quorum-sensing, where gene expression is regulated when a signal reaches a threshold concentration. in many bacteria, qs influences biofilm formation such as shown in p. aeruginosa, where mutants defective in qs form thin and dense biofilm, while the wildtype forms thick biofilms (davies et al. 1998 ). one of the signals that is produced in gram-negative bacteria is n-acylhomoserine lactones (ahls). ahl consists of a lactone ring and an n-acyl side chain whose length ranges from 4 to 20 carbons, which may have additional modifications (arashida et al. 2018) . p. denitrificans produces nhexadecanoyl-l-homoserine lactone (c16-hsl) as its qs signal (schaefer et al. 2002) , which is synthesized by a luxi homolog, pdni ). the pdni mutant strongly aggregates and forms a thick biofilm in comparison to the wildtype, indicating that qs down-regulates aggregation in p. denitrificans (morinaga et al. 2018) . it suggests that c16-hsl regulates eps production, and this relation, once verified, would provide a direct link between qs and biofilm formation. interestingly, p. denitrificans can respond to other ahl signals in different manners and control biofilm formation and cell aggregation (morinaga et al. 2018 ). long-chain ahls (c10 to c18-hsl) inhibit aggregation or biofilm formation similarly to c16-hsl. while p. denitrificans does not respond to short-chain ahls (c4 to c8-hsl) alone, when c4-and c6-hsl are present together with c16-hsl, it can modulate the threshold concentration and less c16-hsl is required to inhibit cell aggregation of the pdni mutant. on the other hand, more c16-hsl is required to inhibit aggregation in the presence of c8 and c10-hsl. such interaction with other signals suggests how this bacterium forms biofilm in a polymicrobial community. the classical qs model is based on the assumption that diffusible qs signals synchronously change target gene expression when the signal concentration reaches a certain threshold (fuqua et al. 1994) . however, hydrophobic signals such as the long-chain ahls, including c16-hsl, cannot freely diffuse in an aquatic environment, and how hydrophobic signals are released and transmitted in the environment had always been a question. in p. denitrificans, it was shown that this bacterium uses membrane vesicles (mvs) to release these signal molecules from the cell and to deliver them to other cells. mvs are produced by most bacteria including gram-negative and gram-positive bacteria and are abundant in the eps matrix of biofilms (schooling and beveridge 2006; toyofuku et al. 2012 toyofuku et al. , 2015 toyofuku et al. , 2018 . the size of mvs ranges from 20 to 400 nm and they carry cellular components such as dna, rna, proteins, and metabolites as their cargo. mvs are involved in various bacterial and host-bacterial interactions and are also used as a platform for therapeutics such as vaccine development (toyofuku et al. 2015) . mvs isolated from p. denitrificans can induce qs targeted gene expression and can also regulate cell aggregation by delivering c16-hsl ). furthermore, one mv contains a much higher amount of c16-hsl than the threshold concentration that would induce gene expression in a p. denitrificans cell. signals packed in mvs would lead to binary signaling, where qs is triggered only in cells that receive mvs, out of the whole cell population. in addition, the calling distance of this digital signaling system would be longer than the free diffusion of signals, as packaging in mvs would prevent the signals from being diluted below the threshold concentration. mvs released from p. denitrificans also show a propensity of cell targeting and have a high affinity to kin species than other species such as p. aeruginosa and p. putida (fig. 2) . mvs can also absorb free long-chain ahls from the environment that can be utilized by p. denitrificans (morinaga et al. 2018) . hence, mvs are involved in trafficking signals that lead to different socioecological consequences. the delivery of hydrophobic signals by mvs is also reported in other bacteria (brameyer et al. 2018; feitosa-junior et al. 2019; mashburn and whiteley 2005) and could be a general way to disperse the signals. p. denitrificans forms a remarkably thin biofilm, which is unique when compared to other bacteria. furthermore, in many bacteria, cyclic-di-gmp and qs positively regulate biofilm formation, while these factors inhibit biofilm formation in p. denitrificans. disruption of qs system leads to thick biofilm formation, indicating that this bacterium possesses the ability to form thick biofilms while repressing it at normal growth conditions (morinaga et al. 2018) . further understanding of environmental factors that control cyclic-di-gmp levels and qs, together with identification of qs-regulated biofilm formation factors, could be key in understanding biofilm formation. in addition, bapa protein, the key factor in the surface attachment of this bacterium, alters the hydrophobicity of the cells, and it would be of interest to examine the influence of bapa expression on mv delivery and c16-hsl signaling in biofilms. one of the advantages of keeping the biofilm thin is that each cell in the biofilm has more access to nutrients than in thick biofilms. however, this aspect is yet to be explored in detail. biofilm thickness is a key property of its function (suarez et al. 2019) , and optimizing the uptake of nutrients by biofilms could be applied to industries including wastewater treatment process using biofilms. p. denitrificans has great potentials in its application for biotechnology, and understanding its biofilm formation may ultimately lead to optimize its functionality. identification of novel long chain n-acylhomoserine lactones of chain length c20 from the marine phototrophic bacterium rhodovulum sulfidophilum nitric oxide regulation of bacterial biofilms nitric oxide signaling in pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic di-gmp levels, and enhanced dispersal molecular architecture and assembly principles of vibrio cholerae biofilms outer 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secretion systemdependent adhesin bapa remediation of pharmaceuticals and personal care products using an aerobic granular sludge sequencing bioreactor and microbial community profiling using solexa sequencing technology analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations bioproduction research institute, national institute of advanced industrial science and technology, central 6, 1-1-1 higashi, tsukuba, ibaraki 305-8566, japan 2 bioproduction research institute, national institute of advanced industrial science and technology, 2-17-2-1, tsukisamu-higashi, toyohira-ku, sapporo, japan acknowledgments mt was supported by a grant-in-aid for scientific research (19h02866 and 19h05682) from the ministry of education, culture, sports, science and technology of japan (mext). nn was supported by the japan science and technology agency, erato (jpmjer1502) and grant-in-aid for scientific research (19h05682 and 16h06382) from mext. conflict of interest the authors declare that they have no conflict of interest.ethical approval this article does not contain any studies with human participants or animals by any of the authors. key: cord-012086-sqv56mmq authors: meijnen, jean-paul; verhoef, suzanne; briedjlal, ashwin a.; de winde, johannes h.; ruijssenaars, harald j. title: improved p-hydroxybenzoate production by engineered pseudomonas putida s12 by using a mixed-substrate feeding strategy date: 2011-02-02 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3089-6 sha: doc_id: 12086 cord_uid: sqv56mmq the key precursors for p-hydroxybenzoate production by engineered pseudomonas putida s12 are phosphoenolpyruvate (pep) and erythrose-4-phosphate (e4p), for which the pentose phosphate (pp) pathway is an important source. since pp pathway fluxes are typically low in pseudomonads, e4p and pep availability is a likely bottleneck for aromatics production which may be alleviated by stimulating pp pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. as p. putida s12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from e. coli was introduced into the p-hydroxybenzoate producing strain p. putida s12palb2. the initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated pp pathway activity. xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 cmol% (0.1 g p-hydroxybenzoate/g substrate). the yield improvements were most pronounced with glycerol, which probably related to the availability of the pep precursor glyceraldehyde-3-phosphate (gap). thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol. pseudomonas putida s12 is a solvent-tolerant bacterium that has been developed as a platform host for the production of a range of substituted aromatic compounds such as phenol, tcinnamate, p-coumarate, p-hydroxybenzoate, and p-hydroxystyrene (nijkamp et al. 2005; nijkamp et al. 2007; verhoef et al. 2007; verhoef et al. 2009; wierckx et al. 2005) . its solvent tolerance properties enable p. putida s12 to produce these toxic hydrophobic compounds to high titres without provoking harmful effects (de bont 1998) . furthermore, in situ product extraction can be applied in fermentations by adding a second phase of a water-immiscible solvent, preventing the accumulation of product to concentrations that are inhibitory even to solvent-tolerant microorganisms (heipieper et al. 2007; verhoef et al. 2009 ). the production of aromatic compounds by engineered p. putida s12 is based on the conversion of endogenously formed tyrosine or phenylalanine. the key precursors of these aromatic amino acids are phosphoenolpyruvate (pep) and erythrose-4-phosphate (e4p) (fig. 1) . pep is produced in the lower glycolysis from glyceraldehyde-3-phosphate (gap). gap is formed from glucose, either via the entner-doudoroff pathway or via the pentose phosphate (pp) pathway, whereas e4p is derived exclusively from the pp pathway. in view of the typically low activity of the pp pathway in p. putida (del castillo et al. 2007; fuhrer et al. 2005; wierckx et al. 2009 ), the availability of e4p and pep may present a bottleneck for efficient aromatics production. increasing the availability of e4p and pep was therefore expected to enhance the production of aromatic compounds by engineered p. putida s12, as previously demonstrated for the pre-aromatic compounds chorismate and shikimate in escherichia coli (martinez et al. 2008) . the availability of pep and e4p may be improved by stimulating pp pathway fluxes through pentose (co-) feeding, as was demonstrated previously in e. coli (gonzalez et al. 2002) . unfortunately, this strategy cannot be applied to p. putida s12 as this strain lacks the natural ability to utilize pentoses. however, in previous work, we successfully introduced xylose utilization, via the xylose isomerase and pp pathway, into wild-type p. putida s12 (meijnen et al. 2008 ). in the present study, a similar approach was employed to introduce xylose catabolism into p. putida s12palb2. this p. putida s12-derived strain produces p-hydroxybenzoate, which was selected as a model value-added aromatic compound derived from the aromatic amino acid biosynthesis pathway (verhoef et al. 2010) . the effect of xylose co-feeding on p-hydroxybenzoate production was assessed using glucose as the primary fig. 1 schematic representation of the biosynthetic pathways for phydroxybenzoate production from glycerol, glucose, and xylose. the scheme shows only the relevant routes. heterologous genes are indicated in italics and underlined. xylose isomerase (xyla); xylulokinase (xylb); phenylalanine/tyrosine ammonia lyase (pal/ tal). glucose-6-phosphate (g6p); fructose-6-phosphate (f6p); fructose-1,6-bisphosphate (f1,6bp); triose-3-phosphate (t3p); phosphoenolpyruvate (pep); pyruvate (pyr); glycerol-3-phosphate (gly3p); ribulose-5phosphate (ru5p); xylulose-5phosphate (xu5p); ribose-5phosphate (r5p); glyceraldehyde-3-phosphate (gap); sedoheptulose-7-phosphate (s7p); erythrose-4-phosphate (e4p); 3deoxy-d-arabino-heptulosonate-7phosphate (dahp); chorismate (cho); phenylalanine (phe); cinnamate (cin); tyrosine (tyr); p-coumarate (coum); 4hydroxyphenylpyruvate degradation pathway (hd pathway); protocatechuate degradation pathway (pd pathway) carbon source, mimicking lignocellulosic hydrolysate that typically contains high levels of both glucose and xylose. alternatively, glycerol was employed as primary carbon source, being a good source for pep as well as being a model for raw glycerol waste from biodiesel production. bacterial strains, plasmids, and culture conditions the strains and plasmids used in this study are listed in table 1 . the media used were luria broth (lb) (sambrook et al. 1982 ) and a phosphate-buffered minimal medium (mm; (verhoef et al. 2010) ). in minimal media, 12 mm of xylose (mmx), 10 mm of glucose (mmg), or 20 mm of glycerol (mmgly) was used as sole carbon source, unless stated otherwise. antibiotics were added as required to the media to the following final concentrations: gentamicin, 10 μg ml −1 (mm) or 25 μg ml −1 (lb); tetracycline, 10 μg ml −1 (e. coli), 40 μg ml −1 (p. putida s12 in mm) or 60 μg ml −1 (p. putida s12 in lb). in view of the photosensitivity of tetracycline, amber bottles and light tight fermentors were employed for culturing. the expression of an additional copy of the native arof-1 gene (wierckx et al. 2005) as well as the expression of the xylab_fgh genes from e. coli (both under the control of the nagaa promoter) were induced by addition of 0.1 mm of sodium salicylate. batch experiments were performed in boston bottles containing 20 ml of minimal medium in a horizontally shaking incubator at 30°c. cultures were inoculated to a starting optical density at 600 nm (od 600 ) of 0.1 with cells precultured on mm with xylose, glucose, or glycerol, depending on the carbon source to be used. carbon-limited chemostat cultivations were performed as described previously (verhoef et al. 2007 ) in 1 l fermentors with a bioflo110 controller (new brunswick scientific) using mm containing different mixtures of glycerol and xylose or glucose and xylose (total carbon (c) concentration, 60 c mm), 10 μg ml −1 gentamicin, 40 μg ml −1 tetracycline, and 0.1 mm sodium salicylate. chemostats were inoculated with a 35-ml inoculum of a late-log phase preculture on mmx. for chemostat cultivations on xylose as sole carbon source, the dilution rate (d) was set at 0.05 h −1 since wash-out occurred at higher dilution rates. for glycerol-xylose mixtures, the d was initially set to 0.05 h −1 . when steady state was reached, the cultures were sampled and d was increased to 0.1 h −1 . (hartmans et al. 1990 ) p. putida s12b2 poba and hpd knockout strain with an enhanced flux towards tyrosine (verhoef et al. 2010 ) p. putida s12palb2 p. putida s12b2 containing plasmid pjt'tpal (verhoef et al. 2010 ) p. putida s12b6 gcd knockout strain derived from p. putida s12b2 this study p. putida s12palb6 p. putida s12b6 containing plasmid pbt'tpal this study p. putida s12xylb6 p. putida s12b6 containing plasmid pjntxylab_fgh this study p. putida s12xylb7 p. putida s12xylb6 evolved to efficient xylose utilizer this study p. putida s12pal_xylb7 p. putida s12xylb7 containing plasmid pbt'tpal this study escherichia coli dh5α supe44 δlacu169 (φ80 laczδm15) hsdr17 reca1 enda1 gyra96 thi-1 rela1 upon reaching steady state at d=0.1 h −1 , the cultures were sampled again. for glucose-xylose mixtures, the d was set at 0.1 h −1 . the cultures were considered to be at steady state when no significant changes were measured in cell density, stirring speed, and p-hydroxybenzoate concentration after at least five volume changes at the corresponding d. optical densities were measured at 600 nm (od 600 ) using an ultrospec cell density meter (ge healthcare). an optical density of 1.0 corresponds to a cell dry weight (cdw) of 0.49 g l −1 . p-hydroxybenzoate was analyzed by hplc (agilent 1100 system) using a zorbax 3.5 μm sb-c18 column (4.6×50 mm) and a diode-array detector set at 254 nm. as the eluent, a linear gradient of acetonitrile in kh 2 po 4 -buffer (50 mm, ph 2, 1% acetonitrile) was used, increasing from 0% to 25% in 4.9 min at a flow of 1.5 ml min −1 . glucose, xylose, and glycerol were analyzed with a dionex ics3000 system as described previously (meijnen et al. 2008; verhoef et al. 2010 for constructing plasmid pbt'tmcs (tc r ) the tac expression cassette and chloramphenicol (cm) marker of pjt'tmcs (verhoef et al. 2010) were amplified by pcr using primers 1 and 2 ( table 2 ). the resulting pcr product was digested with kpn2i and xmaji (restriction sites present in the amplified fragment) and consequently ligated in a kpn2i-xbai (compatible with xmaji) digested pbbr1mcs vector, yielding pbt'tmcs (cm r ). the cm marker was replaced by a tetracycline (tc) marker, which was obtained by pcr using primers 3 and 4 (table 2 ) on vector pto1 (kieboom and de bont 2001) and cloned into the pagi and ncoi restriction sites of pbt'tmcs (cm r ), yielding pbt'tmcs (tc r ). for constructing pbt'tpal, the pal gene from pjttpal (verhoef et al. 2007 ) was obtained as a kpni-noti fragment and purified from agarose gel. the purified fragment was ligated into kpni-noti-digested pbt'tmcs, yielding pbt'tpal. expression vector pjntxylab_fgh was constructed by cloning xylab and xylfgh in vector pjntmcs (meijnen et al. 2008) . the genes were amplified by pcr using genomic dna from e. coli dh5α as the template and oligonucleotide primers 5-8 ( table 2 ). the resulting fragments were ligated into vector pjntmcs using the restriction sites kpni and noti for xylab, and nhei and sfii for xylfgh. the resulting plasmid was designated pjntxylab_fgh. the gcd knockout mutant of p. putida s12b2 (verhoef et al. 2010 ) was constructed in analogy to the gcd knockout mutant of wild-type p. putida s12 (meijnen et al. 2008 ). the gene replacement plasmid for the gcd gene, pjqgcd:: tetaloxp was constructed from the suicide vector pjq200sk (quandt and hynes 1993) based on the pjqgcd::kana vector (meijnen et al. 2008) . the loxp-kanar-loxp fragment, encoding kanamycin resistance in vector pjqgcd::kana was replaced by the loxp-teta-loxp fragment (sauer and henderson 1988; sternberg and hamilton 1981) , coding for tetracycline resistance, using xbai. construction of a xylose-utilizing p-hydroxybenzoateproducing strain in order to establish p-hydroxybenzoate production from xylose, the optimized base strain for p-hydroxybenzoate production, p. putida s12b2 (table 1 ; (verhoef et al. 2010) ), was engineered for xylose utilization. first, the gcd gene encoding glucose dehydrogenase was disrupted in order to eliminate xylose oxidation, which makes xylose effectively unavailable for the xylose isomerase pathway (meijnen et al. 2008 ). subsequently, the xylab_fgh genes from e. coli dh5α (encoding xylose isomerase, xylulokinase, and a high-affinity xylose transporter) were introduced. the resulting strain, p. putida s12xylb6, showed very slow growth on minimal medium with xylose as the sole carbon source, requiring 16 days to reach a cell dry weight (cdw) of 0.69 g l −1 . this result was in agreement with previous findings, and evolutionary selection was performed to improve xylose utilization as described previously (meijnen et al. 2008) . after four transfers, p. putida s12xylb7 was obtained that exhibited a maximum growth rate of 0.34 h −1 on xylose and a biomass-tosubstrate yield (y xs ) of 51 cmol%. the phenylalanine/tyrosine ammonia lyase (pal/tal) expression plasmid pbt'tpal was introduced into p. putida s12xylb7 to establish p-hydroxybenzoate production, yielding strain p. putida s12pal_xylb7. remarkably, upon introduction of pbt'tpal, the maximum growth rate on xylose was reduced by a factor 3 compared to the plasmid-free strain, whereas the growth rate on glucose was not affected (results not shown). still, p-hydroxybenzoate was efficiently produced from xylose in batch cultivations, at a product-to-substrate yield (y ps ) of 12.4 cmol% (table 3) . another remarkable observation was the apparently improved p-hydroxybenzoate yield after evolutionary selection. compared to the non-evolved strain p. putida s12palb6, the product-to-substrate yield of strain s12xyl_palb7 on glucose had increased from 14.2 to 17.4 cmol%. on glycerol, the product-to-substrate yield improved from 15.4 to 19.3 cmol% (data not shown, respectively, table 3 ). in previous work on the engineered xylose-utilizing p. putida strain s12xylab2, a diauxic shift was observed in batch cultivations on mixtures of glucose and xylose, glucose being the preferred carbon source (meijnen et al. 2008) . obviously, the occurrence of diauxy would invalidate the concept of co-feeding two substrates simultaneously to different pathways, in order to improve the availability of two key precursors. therefore, a chemostat cultivation setup was selected for the mixed-substrate experiments, with glucose or glycerol as the limiting nutrient. varying amounts of xylose were used (0% to 75% of 60 mm total carbon) to replace the primary carbon source. although the primary carbon source was completely metabolized as expected, residual xylose was observed in the chemostat effluent. the extent to which xylose was utilized was furthermore very different for glucose and glycerol (fig. 2) . on glycerol-xylose mixtures, the amount of xylose consumed correlated well with the amount of xylose in the feed. the residual xylose observed was in the same range as in chemostat cultivations with xylose as the sole carbon source (approximately 0.27 mm). however, with glucose as the primary carbon source, the residual xylose concentrations were much higher and, furthermore, increased more than proportionally to the relative xylose concentration in the feed. clearly, the capacity to transport and/or utilize xylose was dependent on the type of primary carbon source as well as on the relative amount of the primary carbon source in the feed. chemostat cultivations with p. putida s12pal_xylb7 on glucose as single carbon source showed a product-tosubstrate yield of 4.9 cmol%, with a specific production (fig. 3b) . the biomass yield was 42.5 cmol% (fig. 3a) . when xylose was co-fed with glucose, the biomass-to-substrate yield slightly increased with increasing xylose concentrations (fig. 3a) . this is in agreement with the higher biomass yield observed on xylose compared to glucose (y xs of 47.7 vs. 42.5 cmol%, fig. 3e, a) . moreover, the production of phydroxybenzoate improved significantly when xylose was co-fed with glucose. the product-to-substrate yield reached a maximum of 7.9 cmol%, and the specific production rate increased by a factor of 1.5 to 12.0 μmol c (g cdw) −1 h −1 (fig. 3b) . these results clearly demonstrated that simultaneous utilization of glucose and xylose was beneficial for phydroxybenzoate production. surprisingly, the amount of xylose that was co-fed did not significantly affect y ps and q p within the range tested (fig. 3b) . in chemostats with glycerol as the sole carbon source, p. putida s12pal_xylb7 produced p-hydroxybenzoate at a yield of 8.5 cmol%. notably, this represents a 1.7-fold improvement over the product yield observed in glucosegrown cultures. also in shake-flasks, an improved phydroxybenzoate yield on glycerol was observed (table 3) , although not as pronounced as in the chemostat cultivations. in addition to the product-to-substrate yield, also the biomass-to-substrate yield was improved 1.23-fold on glycerol (fig. 3c) . because of the improved biomass yield, the effect of the primary substrate on the specific phydroxybenzoate production rate q p was limited: the q p on glycerol (8.57 μmol c (g cdw) −1 h −1 ) was only slightly higher than on glucose (7.96 μmol c (g cdw) −1 h −1 ). the intrinsically improved p-hydroxybenzoate production from glycerol was further enhanced by xylose cofeeding. the product-to-substrate yield increased by a factor 1.9 to a maximum of 16.3 cmol% (fig. 3d) . the q p improved along with y ps , to a maximum value of 18 μmol c (g cdw) −1 h −1 . as observed for the glucosegrown chemostats, the relative amount of xylose in the feed did not affect the efficiency of p-hydroxybenzoate production from glycerol-xylose mixtures (fig. 3d) . production of p-hydroxybenzoate from xylose as single carbon source in order to verify that the improved p-hydroxybenzoate production was a result of the simultaneous utilization of xylose and glucose or glycerol, and not merely of xylose utilization alone, chemostat experiments were performed with p. putida s12pal_xylb7 on xylose as single carbon source. in agreement with the reduced growth rate observed in xylose-grown batch cultures after introducing the pal/tal expression plasmid, p. putida s12pal_xylb7 washed out above a dilution rate of 0.05 h −1 . therefore, chemostats on xylose were operated at a dilution rate of 0.05 h −1 . also chemostats with a mixed glycerol-xylose feed, or glycerol as the sole carbon source, were operated at this dilution rate, as control for growth-rate effects on p-hydroxybenzoate production. biomass and product yields were not significantly different in chemostats operated at d=0.05 h −1 and d= 0.1 h −1 (fig. 3c-f ), whereas the specific production and substrate uptake rates were proportional to the dilution rate as expected (fig. 3e-f) . on xylose as a single carbon source, the product-to-substrate yield (5.6 cmol%) was 1.5fold lower than on glycerol alone (table 3) , which result is in agreement with the product yields observed in shake flask cultures (table 3 ). this demonstrates that the improved p-hydroxybenzoate production is indeed a result from co-feeding two carbon sources. furthermore, as the product yield on mixtures of xylose and glycerol or glucose consistently exceeded the product yield on either individual carbon source (table 3) , it was concluded that co-feeding xylose to glucose or glycerol has a beneficial effect over feeding individual carbon sources for p-hydroxybenzoate production. a mixed-substrate feeding strategy was devised to improve aromatics production by engineered p. putida s12. the approach was based on the assumption that the precursors fig. 2 relative xylose uptake as a function of the xylose fraction in the feed. the solid line represents the theoretical maximum uptake of xylose; the dotted lines represent the actual uptake of xylose with glycerol as co-substrate (triangles) or glucose as co-substrate (circles). data are the average from two independent cultivations; error bars represent the maximum deviation from the mean e4p and pep were limiting factors for aromatic biosynthesis and that their availability could be improved by stimulating the pp pathway fluxes through pentose cofeeding. a p-hydroxybenzoate producing strain, p. putida s12palb2, was selected as an aromatics-producing model system. as this strain does not have the natural ability to utilize pentoses, a xylose isomerase pathway was introduced, and the initially low growth rate on xylose was improved via an evolutionary selection procedure. surprisingly, tenfold less transfers were required to achieve growth characteristics similar to those of the previously evolved xylose-utilizing strain p. putida s12xylab2 (meijnen et al. 2008) . in part, this can be attributed to the targeted disruption of gcd, since more than ten transfers had been required for strain s12xylab2 to acquire a gcd negative phenotype. in addition to the xylose utilization efficiency, also the p-hydroxybenzoate yield was improved after the evolutionary selection. this phenomenon may be explained by the increased pp pathway activity associated with the improved xylose utilization phenotype, leading to an intrinsically improved e4p and pep availability, independently from xylose co-feeding. as anticipated, xylose co-feeding considerably improved the p-hydroxybenzoate yield. the increased product yield was observed with both glucose and glycerol as the primary substrate and was shown not to be caused by xylose consumption per se. remarkably, both the product and biomass yield on glycerol were consistently higher comfig. 3 growth and p-hydroxybenzoate production of p. putida s12pal_xylb7 in chemostat cultivations on various mixtures of carbon sources at various dilution rates (d). the biomassto-substrate yield (y xs ), productto-substrate yield (y ps ), and product-to-biomass yield (y px ) expressed as in cmol biomass or product per cmol substrate or biomass. the specific carbon uptake rate (q s ) and specific p-hydroxybenzoate production rate (q p ) were given in cμmol substrate or product per gram cell dry weight (cdw) per hour. all calculated figures were corrected for the consumed amount of substrate pared to glucose, either with or without xylose co-feeding. this may be attributed to regulatory effects (e.g., carbon catabolite repression) but could also indicate that pep availability is more critical for efficient p-hydroxybenzoate production than e4p availability. if it is assumed that pyruvate dikinase (pep synthase) is active only under gluconeogenic conditions (sauer and eikmanns 2005) , twice the amount of gap (and, thus, pep) can be obtained from glycerol compared to glucose, which is metabolized via the entner-doudoroff pathway in pseudomonads (del castillo et al. 2007; fuhrer et al. 2005) . in addition, the glycerol-associated yield improvement appears to be connected to the evolutionary selection, since no such effect has been observed with the parent strains of p. putida s12pal_xylb7 (verhoef et al. 2010) . presumably, the increased pp pathway activity associated with the efficient xylose-utilizing phenotype may allow for a more efficient equilibration of pep and e4p levels, resulting in more efficient p-hydroxybenzoate production. it should be noted that the applied proportion of xylose in the feed showed little effect within the range tested, whether the primary substrate was glucose or glycerol. apparently, the phydroxybenzoate production is not very sensitive to variations in relative xylose concentrations above a certain threshold value. unexpectedly, the capacity to transport and/or utilize xylose appeared to be dependent on the primary carbon source. with glycerol, a low concentration of residual xylose was observed that is presumably close to the k m of the-yet unidentified-xylose transporter in p. putida s12pal_xylb7. with glucose as the primary substrate, however, the residual xylose concentrations were higher and furthermore increased more than proportionally with increasing amounts of xylose in the feed. although this phenomenon is still subject to further study, it may be hypothesized that xylose transport in p. putida s12pal_-xylb7 is pep dependent. this would be consistent with the observed increase in residual xylose concentrations with decreasing glucose feed (an already relatively inefficient source of pep), the relative independency between residual xylose concentration and glycerol feed (a good source of pep), and the decreased growth rate on xylose when pal/tal was introduced (drain on pep for p-hydroxybenzoate production). in that case, replacing any pep-dependent transport systems would be an obvious target for further strain improvement. the gap/pep availability may furthermore be improved by constructing an ed-negative, glycolytic p. putida s12 strain. the contribution of the (atp-driven) e. coli xylose transporter xylfgh to xylose import was presumably limited as observed previously (meijnen et al. 2008) . we have demonstrated that p-hydroxybenzoate production in p. putida can be considerably improved by cofeeding different carbon sources that are metabolized via different, (partly artificial) pathways. thus, the availability of the key aromatics precursors, pep and e4p, is improved. in addition to p-hydroxybenzoate, the production of other aromatic compounds derived from aromatic amino acids may be stimulated via this strategy. moreover, lignocellulosic hydrolysate, the expected major feedstock for future production of biobased fuels and chemicals (himmel and bayer 2009; kumar et al. 2008; lange 2007) , seems to be ideally suited for aromatics production since glucose and xylose are the predominant constituents. also the improved production on glycerol presents an additional possibility to deploy a cheap and abundant waste substrate for biocatalytic production of (fine) chemicals. solvent-tolerant bacteria in biocatalysis convergent peripheral pathways catalyze initial glucose catabolism in pseudomonas putida: genomic and flux analysis experimental identification and quantification of glucose metabolism in seven bacterial species global gene expression differences associated with changes in glycolytic flux and growth rate in escherichia coli during the fermentation of glucose and xylose bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase solvent-tolerant bacteria for biotransformations in two-phase fermentation systems lignocellulose conversion to biofuels: current challenges, global perspectives identification and molecular characterization of an efflux system involved in pseudomonas putida s12 multidrug resistance bioconversion of lignocellulosic biomass: biochemical and molecular perspectives lignocellulose conversion; an introduction to chemistry, process and economics coutilization of glucose and glycerol enhances the production of aromatic compounds in an escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system engineering pseudomonas putida s12 for efficient utilization of d-xylose and l-arabinose the solvent-tolerant pseudomonas putida s12 as host for the production of cinnamic acid from glucose optimization of the solvent-tolerant pseudomonas putida s12 as host for the production of p-coumarate from glucose versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria molecular cloning. a laboratory manual the pep-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria site-specific dna recombination in mammalian cells by the cre recombinase of bacteriophage p1 bacteriophage p1 site-specific recombination. i. recombination between loxp sites bioproduction of p-hydroxybenzoate from renewable feedstock by solventtolerant pseudomonas putida s12 bioproduction of p-hydroxystyrene from glucose by the solvent-tolerant bacterium pseudomonas putida s12 in a twophase water-decanol fermentation comparative transcriptomics and proteomics of p-hydroxybenzoate producing pseudomonas putida s12: novel responses and implications for strain improvement engineering of solvent-tolerant pseudomonas putida s12 for bioproduction of phenol from glucose metabolic flux analysis of a phenol producing mutant of pseudomonas putida s12: verification and complementation of hypotheses derived from transcriptomics acknowledgements the authors thank karin nijkamp for constructing the basic expression plasmids. this project is financially supported by the netherlands ministry of economic affairs and the b-basic partner organizations (http://www.b-basic.nl) through b-basic, a public-private nwo-acts programme (acts=advanced chemical technologies for sustainability). this project was co-financed by the kluyver centre for genomics of industrial fermentation, which is part of the netherlands genomics initiative/netherlands organization for scientific research.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-282321-svoshzz8 authors: eboigbodin, kevin; filén, sanna; ojalehto, tuomas; brummer, mirko; elf, sonja; pousi, kirsi; hoser, mark title: reverse transcription strand invasion based amplification (rt-siba): a method for rapid detection of influenza a and b date: 2016-04-11 journal: appl microbiol biotechnol doi: 10.1007/s00253-016-7491-y sha: doc_id: 282321 cord_uid: svoshzz8 rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (pcr), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. we previously described a novel isothermal nucleic acid amplification method, ‘strand invasion based amplification’ (siba®), with high analytical sensitivity and specificity, for the detection of dna. in this study, we describe the development of a variant of the siba method, namely, reverse transcription siba (rt-siba), for the rapid detection of viral rna targets. the rt-siba method includes a reverse transcriptase enzyme that allows one-step reverse transcription of rna to complementary dna (cdna) and simultaneous amplification and detection of the cdna by siba under isothermal reaction conditions. the rt-siba method was found to be more sensitive than pcr for the detection of influenza a and b and could detect 100 copies of influenza rna within 15 min. the development of rt-siba will enable rapid and accurate diagnosis of viral rna targets within point-of-care or central laboratory settings. electronic supplementary material: the online version of this article (doi:10.1007/s00253-016-7491-y) contains supplementary material, which is available to authorized users. influenza a and b are among the most common causes of acute human respiratory illness and are associated with high morbidity and mortality rates in infants, the elderly, and immunocompromised individuals (mallia and johnston 2007; simonsen et al. 2000) . today, patients with influenza infection may be treated with antiviral drugs; however, these treatments are only effective when started within the first 2 days of the illness (stiver 2003) . thus, timely and accurate diagnosis of influenza plays an important role in targeting antiviral treatment and, furthermore, has a significant role in the prevention of the misuse of antibiotics, since the symptoms of influenza are often similar to those of other respiratory illnesses caused by bacterial infections (low 2008) . nucleic acid amplification tests (naats) are becoming the method of choice for routine diagnosis of influenza and other respiratory viruses within clinical laboratory settings. naats offer superior sensitivity and specificity over serology, immunoassays, and traditional viral culture-based detection methods (mahony et al. 2011 ). real-time reverse transcription polymerase chain reaction (rt-pcr) assays have been developed for the diagnosis of influenza and remain the most common platform used in naat. the rt-pcr method includes, as a first incubation step, the reverse transcription of rna to complementary dna (cdna) by a transcriptase enzyme, followed by a pcr cycling stage. a rt-pcr run is typically completed within 2 h. most reverse transcriptases are not thermostable; consequently, the reverse transcription step must be performed prior to the pcr step (gerard et al. 2002) . typically, rt-pcr requires the use of heavy and sophisticated sanna filén and tuomas ojalehto contributed equally to this work. electronic supplementary material the online version of this article (doi:10.1007/s00253-016-7491-y) contains supplementary material, which is available to authorized users. thermal cyclers and skilled personnel, which limits its use for in field or point-of-care applications. in an attempt to address this drawback, isothermal nucleic acid amplification platforms have been developed, which do not require the use of heavy, sophisticated thermal cyclers. instead, isothermal nucleic acid amplification methods require only small instruments, enabling naats to be performed within point-of-care settings. this has the potential to allow prompt treatment of patients and limit the inappropriate use of antibiotics and antiviral drugs. we previously described a novel isothermal nucleic acid amplification method, 'strand invasion based amplification' (siba®), with high analytical sensitivity and specificity (hoser et al. 2014) . the method relies on the recombinase-dependent insertion of a single-stranded invasion oligonucleotide (io) into a complementary region of a target duplex dna, which results in the dissociation of the target duplex. this dissociation event generates single-stranded dna, which in turn allows targetspecific primers to bind and extend the target sequence via the action of a dna polymerase. the method was previously found to be useful for the rapid detection of dna from pathogens (hoser et al. 2014) . in this study, we describe the development of a variant of siba technology, namely, reverse transcription siba (rt-siba), for the rapid detection of viral rna targets. we demonstrate the efficacy of rt-siba in the detection of viral rna by developing rt-siba assays for both influenza a and b. the assays were designed to detect sequences within conserved regions of the influenza genome. the rt-siba method includes a reverse transcriptase enzyme that allows a one-step reverse transcription of rna to cdna and simultaneous amplification and detection of the cdna with siba under isothermal reaction conditions. furthermore, we compared the performance of rt-siba with the previously published centers for disease control and prevention (cdc) real-time pcr protocol for the detection of influenza a and b. the oligonucleotides used in this study were purchased from eurofins genomics (ebersberg, germany) or integrated dna technologies (leuven, belgium) and purified by the manufacturer using either reverse-phase hplc (for primers and probes) or page (for the ios). oligonucleotide sequences are shown in fig. 1 . amplirun purified and quantified rna from influenza a (h1n1, h3n2, and h5n1) and b were obtained from vircell (granada, spain) and used to determine the analytical sensitivity of both the rt-siba and rt-qpcr methods. nattrol flu verification panel (zeptometrix, buffalo, ny, usa) samples were used to establish the ability of rt-siba to detect influenza viral particles in the presence of a simulated clinical sample matrix. each pathogen was transferred using nasopharyngeal swabs into a lysis buffer (10 % triton x-100, sigma-aldrich, usa) and 2 μl was then added to the reaction. a 300-bp template sequence containing the region 2023-2322 from the influenza a virus segment 1 polymerase pb2 gene (kj942711) was inserted into the pexa2 vector by a commercial company (eurofins genomics, ebersberg, germany). the sequence was flanked by a 20-bp t7 promoter sequence and a smai site at the 5′ and 3′ ends, respectively. transcripts were prepared using an in vitro transcription kit (hiscribe™ t7 high yield rna synthesis kit; new england biolabs, ipswich, ma, usa). transcripts were quantified both spectrophotometrically and by quantitative real-time pcr and were used to determine the performance and analytical sensitivity of the influenza a rt-siba assay. the influenza research database (http://www.fludb.org) was used to aid the design of the influenza rt-siba assays (squires et al. 2012) . to design an assay for influenza a, a total of 4575 non-duplicate influenza a segment 1 sequences that had been submitted to the database during the years 2010-2015 were retrieved and aligned. special emphasis was put on the most clinically relevant subtypes, h1n1, h3n2, and h5n1; thus, sequences representing these subtypes were included in the alignment. the alignment was analyzed to find areas of limited variability using jalview (http://www.jalview.org). a highly conserved area of approximately 80 nucleotides was identified at the 3′ end of the aligned segment 1, with a suitable consensus sequence, and this area was selected as the target area for rt-siba assay design. analogously, for the influenza b assay, an alignment of 1495 non-duplicate segment 3 sequences was produced. a highly conserved, approximately 80 nucleotide area was identified at the far 5′ end of the alignment and selected as the assay target area. alignments of the consensus sequences for influenza a and b with the oligonucleotides designed for the siba influenza assays are shown in fig. 1 . in essence, siba amplification is highly specific; therefore, special care was taken to ensure that the majority of any sequence variation fell inside the forward or reverse primers sequences and was limited to a maximum of 1-2 nucleotide changes in the io region (see hoser et al. (hoser et al. 2014 ) with respect to general assay design, siba specificity, and initial screening of suitable oligo combinations). after the assays were established, they were subjected to several rounds of optimization as described below. rt-siba reactions were performed using a commercial siba reagent kit (orion diagnostica oy, espoo, finland) with the addition of 16 u of goscript™ reverse transcriptase (promega, madison, usa). uvsx and gp32 enzymes were each used at 0.25 mg/ml, and the reactions were started using 10 mm magnesium acetate. for the siba influenza a assay, the forward and reverse primers and the io were each used at final concentrations of 400 nm. for the siba influenza b assay, the forward and reverse primers and the io were each used at final concentrations of 200 nm. siba amplification was detected using sybr green 1 (dilution, 1:100,000; thermo fisher scientific, usa). rt-siba reactions were incubated at 41°c for 60 min, and fluorescence readings were taken at 60-s intervals on an agilent mx pro 3005p instrument (agilent technologies, inc., ca, usa). after incubation for 60 min, the instrument was set to run a melt curve from 40 to 95°c, to further assess the specificity of the siba reactions. siba reactions were optimized for the rapid detection of influenza a by first determining the best nucleotide configuration for the io to give the shortest detection time. this was achieved by varying the length and composition of the 5′ nonhomologous (seeding) region of the io. six different ios with seeding regions of lengths varying from 0 to 17 nucleotides were tested. in another approach, the length of the seeding region was maintained at ten nucleotides but the nucleotide composition of the seeding region was modified to contain either polyc, polyt, polya, polyg, poly purines, or poly pyrimidines. the exact sequences of each tested io are listed in table s1 . the performance of each io at 200 nm was assessed by its ability to facilitate amplification of 10,000 copies of in vitro transcribed influenza a rna in the presence of the primers. the best performing seeding region was then chosen for subsequent siba influenza a experiments and adopted for influenza b siba assay design. the performance of rt-siba assays for the detection of influenza a and b was compared with the cdc protocol for real-time rt-pcr of influenza a and b ((who) 30 april 2009; selvaraju and selvarangan 2010). briefly, the rt-pcr reactions were performed with the express one-step superscript® qrt-pcr supermix kit, with rox reference dye (thermo fisher scientific, usa). this kit contains a heat-labile form of uracil dna glycosylase (udg), in addition to superscript® iii reverse transcriptase and platinum® taq dna polymerase, in order to prevent any potential carryover contamination. the reactions were detected using an applied biosystems 7500 pcr instrument (thermo fisher scientific, usa). all primers and probes were used at 1 μm and 200 nm, respectively. the following thermal cycling protocol was used: 50°c for 30 min (cdna synthesis), 95°c for 2 min (reverse transcription and udg inactivation), and 45 cycles of 95°c for 15 s and 55°c for 30 s (pcr amplification). the mechanism of rt-siba is described in fig. 2 . the io, used in siba reactions to separate the target duplex dna, includes a 5′ region that is not homologous to the target sequence. this nonhomologous region, termed the seeding region, is included in the io to allow for optimal coating of the homologous portion by the recombinase. the affinity of the recombinase for dna molecules was previously suggested to be dependent on both the length and nucleotide composition of the target molecule (formosa and alberts 1986) . therefore, we hypothesized that the length and composition of the seeding region could also impact the speed of siba assays. in order to test our hypothesis, siba influenza a assay ios containing seeding regions that differed in length and nucleotide composition were designed and tested, and the results are depicted in fig. 3 . the use of an influenza a assay io with no seeding region resulted in reactions with the longest detection time, suggesting that the seeding region is of vital importance for efficient amplification of the target dna. furthermore, shorter detection times were observed as the length of the seeding region increased. our experiments demonstrate that the optimal length of the seeding region, in terms of assay speed, is a minimum of six nucleotides. to evaluate the impact of the nucleotide composition of the seeding region on assay speed, seven ios with seeding regions containing either polyc, polyt, polya, polyg, poly purines, poly pyrimidines, or mixed nucleotides were tested. the use of io seeding regions containing polyc and polyt resulted in the shortest detection times, while the use of an io containing a polyg seeding region led to the longest detection time. the io containing a seeding region with a mixture of pyrimidine nucleotides was found to display shorter detection times than that with a mixture of purine nucleotides. in fig. 2 a mechanistic description of the rt-siba reaction. 1 rna is reverse transcribed to cdna by the transcriptase enzyme. 2 the siba reaction requires two targetspecific primers and an invasion oligonucleotide (io). all singlestranded elements are coated with gp32. 3 t4 uvsx recombinase polymerizes the io displacing bound gp32. the lengths of the primers used are too short to act as substrates for uvsx. 4 io invades the complementary region of the target duplex through the activity of uvsx. the invasion process facilitates the complete separation of the target duplex, allowing target-specific primers to bind the target. 5 the strand displacement polymerase is able to extend the dissociated target duplex from the primers. this event leads to the production of two copies of the target duplex. the recombinasemediated target duplex separation and polymerase-mediated extension are the basis for exponential amplification conclusion, ios for both influenza a and b assays used in the following experiments were designed to contain seeding regions consisting of a polyc sequence of 10 and 14 nucleotides in length, respectively. cons-b 5' tccagactacaataatacaaaaggccaaaaacacaatggcagaatttagtgaagatcctgaattacaaccagcaatgct 3' io-b 5' ccccccccccccccaaggccaaaaacacaatggcagaatttagugaagauccuga 3' ifb-f 5' actacaataatacaaaag 3' ifb-r 3' ggacttaatgttggtcgt 5' the sensitivities of both the influenza a and b rt-siba assays were assessed against those of the equivalent previously published cdc real-time rt-pcr assays. serial dilutions of quantified amplirun rna controls for influenza a h1n1, h3n2, and h5n1 subtypes, as well as influenza b, were used as templates for both rt-siba and rt-pcr assays. quadruplicate reactions were performed in at least three independent experiments. the diluted samples were analyzed simultaneously with both rt-siba and rt-pcr reactions in order to facilitate comparison between the two methods. the results are shown in fig. 4 and table 1. rt-siba reliably detected as low as ten copies of viral rna per reaction. serial dilutions from 1000 to 10 copies of purified rna from different influenza subtypes showed reproducible and specific amplification when detected using sybr green i fluorescent dye. post-amplification melt curve analysis was performed after each run, and amplification of a single-specific amplicon was detected for both influenza a and b assays (data not shown). furthermore, the influenza a rt-siba assay did not amplify influenza b rna or vice versa. comparison of analytical sensitivity in terms of rna copy numbers per reaction indicated that the rt-siba assays had comparable or superior sensitivity, compared with rt-pcr assays (table 1 ). notably, we were able to demonstrate the sensitivity of rt-siba to be ten copies of target rna per reaction for both influenza a and b assays; however, our experiments demonstrated that, unlike that of the rt-siba assays, the sensitivity of rt-pcr varied depending on the assay and influenza template subtype used. for influenza b assays, rt-siba and rt-pcr assays fig. 3 the impact of changes to the invasion oligonucleotide (io) seeding region nucleotide composition on the average detection time for the siba influenza a assay. the impact of the seeding region a length and b composition (for a ten nucleotide seeding region). the results are presented as the average detection time from eight replicate assays (standard deviation included). the detection time reported was the time at which the probe fluorescent signal exceeded the background signal performed equally well and were both able to successfully amplify ten copies of rna per reaction. however, a significant difference between rt-siba and rt-pcr was observed for the influenza a assays. ten copies of all tested subtypes of influenza a were reproducibly amplified and detected by rt-siba, whereas the rt-pcr assay was only capable of amplifying 100 or 1000 copies of rna per reaction. replicate reactions. the total copy number of template rna per reaction was 10-1000 copies/reaction. the results indicated that rt-siba could be used to reproducibly detect as few as ten copies of influenza rna per reaction. ntc = no template control the average time to achievement of a positive reaction, corresponding to the threshold cycle (ct)-value for rt-pcr reactions, was calculated according to the one-step rt-pcr cycling program, without taking into account the ramping time needed for pcr cycling. rt-siba reactions were performed at a constant temperature, and data were collected at 1 min intervals. therefore, the ct-values of rt-siba reactions corresponded directly to the time required to achieve a positive result. the average times to positive results for both rt-siba and rt-pcr reactions are listed in table 1 . positive results from 100 copies of influenza b rna template per reaction were obtained in an average of 12 min with the rt-siba assay, whereas an average of 53 min was required to yield a positive result by rt-pcr. similarly, 100 copies of influenza a h1n1, h3n2, and h5n1 subtypes were detected by rt-siba in 15, 14, and 12 min, respectively, whereas reaction times of more than 50 min were needed for rt-pcr. these results demonstrate a major advantage of rt-siba over rt-pcr. the specificities of the siba influenza a and b assays were elucidated by challenging the reactions with nucleic acid from six other common human respiratory pathogens. these included human rhinovirus, respiratory syncytial virus (rsv), coronavirus, adenovirus, staphylococcus aureus, and streptococcus pyogenes. all non-influenza nucleic acid templates gave negative results with both the influenza a and b rt-siba assays (data not shown). the influenza a assay was further challenged with an influenza b rna template, which was not detected by the assay. similarly, the influenza b assay did not detect influenza a template rna. these findings suggest that both of the siba influenza assays are specific for their respective targets. the performance of both the influenza a and b rt-siba assays was further evaluated using commercial nattrol flu verification panel samples that contain viral or bacterial particles in a protein matrix, to mimic clinical specimen samples. table 2 . the influenza a rt-siba assay detected all six influenza a strains and did not cross-react with influenza b strains, nor the other respiratory pathogens. similarly, the influenza b rt-siba assay detected the two influenza b strains and did not cross-react with influenza a strains, nor the other respiratory pathogens. this indicates that the influenza a and b rt-siba assays are likely to be suitable for detecting influenza viruses from clinical specimens. in this study, we demonstrated the ability of siba technology to detect rna-pathogens, rt-siba, for the rapid detection of influenza a and b with high analytical sensitivity and specificity. previously, it was demonstrated that siba technology is a feasible method for the reliable detection of dna from pathogenic micro-organisms (hoser et al. 2014) . in this paper, we demonstrate that rt-siba can be applied for the rapid detection of pathogens that have rna, rather than dna, as their genetic material, as is the case for the majority of viruses. the method described here includes a reverse transcriptase enzyme in the same reaction tubes as the other siba reaction components required for nucleic acid amplification and consequently permits a one-step reverse transcription of rna to cdna and simultaneous amplification and real-time detection of cdna by siba. the siba method consists of a recombinase, required for homologous recombination, an io, which is a substrate for the recombinase, and two target-specific primers that are not substrates for the recombinase. the io separates the target duplex, with the aid of the recombinase, consequently allowing the primers to bind and extend the target via the action of a dna polymerase. repeated cycles of duplex separation and primer extension are the basis for exponential amplification. in this report, the amplification times for the siba influenza assays were first optimized by varying the length and composition of the io non-homologous region (i.e., the seeding region). our experiments showed that a polyc or polyt non-homologous io region of at least ten nucleotides in length resulted in the shortest detection times. the recombinase, uvsx binds preferentially to pyrimidine rich nucleotides (formosa and alberts 1986) , however, the reaction demands both the binding and release of the recombinase and consequently the optimal configuration of the seeding region was determined empirically. the necessity of the seeding region demonstrated that the recombinase perform less efficiently at the initial potential binding sites prior to its polymerization from 5′-3′. the average times to detection for both the influenza a and b rt-siba assays were below 15 min for 100 copies of rna. thus, the complete test can be conducted in less than 30 min. this is significantly faster than the real-time rt-pcr method, which took over 1 h for the detection of influenza. in rt-siba, the reverse transcription and cdna amplification occur simultaneously and at the same reaction temperature, thus facilitating rapid detection of target rna. by contrast, in real-time rt-pcr, reverse transcription requires an initial incubation period of 30 min at a constant temperature prior to the repeated pcr cycles of denaturation, annealing, and elongation, resulting in a total run time of 90-120 min. furthermore, rt-sibawas found to be more sensitive than rt-pcr for the detection of influenza a and b. rt-siba displayed a 100-fold improvement in sensitivity, compared with rt-pcr, for the detection for h1n1. the siba reaction components include the single strand binding protein, gp32, which acts as a recombinase accessory protein and is required for the removal of secondary structures within the oligonucleotides used in the assay. it is possible that the presence of gp32 in the rt-siba reaction mixture could also contribute to the high analytical sensitivity displayed by rt-siba for rna targets. this is supported by previous studies, which showed that single binding proteins enhance the yield of cdna synthesis by reverse transcriptase, thus improving dna amplification sensitivity (jefferies and farquharson 2002; villalva et al. 2001) . furthermore, the difference in rt enzymes used in rt-siba and rt-pcr could account for the significant difference in sensitivity between the methods. several studies show that commercially available reverse transcriptases display varying performance regarding cdna yield and sensitivity (levesque-sergerie et al. 2007; okello et al. 2010; ståhlberg et al. 2004) . the performance of rt-siba for the detection of influenza a and b was determined using the commercially available clinical nattrol flu verification panel samples, which simulate clinical specimen samples. the rt-siba influenza assays amplified the influenza specimens specifically, with no cross-reactions with other respiratory pathogens. since rt-siba was found to be less susceptible to sample derived inhibition, the method does not require highly purified rna template material and crude disruption of viral particles using detergents was found to be sufficient. the sensitive and rapid detection of influenza viruses within 15 min, as well as the tolerance to sample derived inhibition, demonstrate that these assays are powerful molecular diagnostics tools. since the method is performed at a low and constant temperature, there is potential for it to be run using battery operated and portable devices of considerably lower complexity than those used for rt-pcr. isothermal methods can reasonably be expected to extend the use of molecular methods outside of centralized laboratory spaces into lower complexity settings, such as smaller laboratories, the field, or even domestic homes. compliance with ethical standards this article does not contain any studies with human participants or animals performed by any of the authors. purification and characterization of the t4 bacteriophage uvsx protein the role of template-primer in protection of reverse transcriptase from thermal inactivation strand invasion based amplification (siba®): a novel isothermal dna amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte effects of choice of reversetranscriptase enzyme and use of t4 gene 32 protein on banding patterns in agarose gel differential display detection limits of several commercial reverse transcriptase enzymes: impact on the low-and high-abundance transcript levels assessed by quantitative rt-pcr reducing antibiotic use in influenza: challenges and rewards molecular diagnosis of respiratory virus infections influenza infection and copd quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored hiv rna evaluation of three influenza a and b real-time reverse transcription-pcr assays and a new 2009 h1n1 assay for detection of influenza viruses the impact of influenza epidemics on hospitalizations influenza research database: an integrated bioinformatics resource for influenza research and surveillance comparison of reverse transcriptases in gene expression analysis the treatment of influenza with antiviral drugs increased yield of pcr products by addition of t4 gene 32 protein to the smart pcr cdna synthesis system cdc protocol of real-time rt-pcr for influenza h1n1. world health organization open access this article is distributed under the terms of the creative comm ons attribution 4.0 international license (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord-004592-a3k56ulv authors: liu, xiaoju; chen, yubin; wu, xiaoping; li, haiyan; jiang, chao; tian, haishan; tang, lu; wang, dezhong; yu, ting; li, xiaokun title: sumo fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (fgf23) date: 2012-01-17 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3864-4 sha: doc_id: 4592 cord_uid: a3k56ulv as a key humoral regulator of phosphate homeostasis and its involvement in the pathogenesis of human disease, human fibroblast growth factor 23 (hfgf23) has become a particularly attractive therapeutic target. to prepare soluble and bioactive recombinant human fgf23 to meet the increasing demand in its pharmacological application, small ubiquitin-related modifier (sumo)-fgf23 fusion gene and fgf23 non-fusion gene were amplified by standard pcr methods and cloned into vector pet-22b and pet-3c, then transformed into escherichia coli rosetta (de3) and bl21 (de3). the best combination of plasmid and host strain was screened, and only rosetta (de3)/pet-sumo-fgf23 was screened for rhfgf23 protein expressed. the average bacterial yield and the soluble expression level of recombinant hfgf23 of three batches attained 687 ± 18 g and 30 ± 1.5%, respectively, after treatment with 0.4 mm isopropyl-thio-β-galactopyranoside for 19 h at 16 °c in a 30-l fermentor, after which it was purified by deae sepharose ff and nickel nitrilotriacetic acid affinity chromatography. once cleaved by the sumo protease, the recombinant human fgf23 was released from the fusion protein. the purity of rfgf23 was shown by high performance liquid chromatography to be greater than 90% and the yield was 60 ± 1.5 mg/l. in vitro data showed that the purified rfgf23 can induce the phosphorylation of mitogen-activated protein kinases in the glioma u251 cell. the results of in vivo animal experiments also showed that rfgf23 could decrease the concentration in the plasma of normal rats fed with a fixed formula diet. electronic supplementary material: the online version of this article (doi:10.1007/s00253-011-3864-4) contains supplementary material, which is available to authorized users. human fibroblast growth factor 23 (hfgf23), a member of the fgf superfamily, was first identified in 2000 (yamashita et al. 2000) . the full-length hfgf23 consists of 251 amino acids with a signal peptide of 24 amino acids at the nterminus, splicing a mature fgf23 polypeptide of 227 amino acids (yamashita et al. 2000) . fgf23 reduces serum phosphate by inhibiting both proximal tubular phosphate reabsorption and intestinal phosphate absorption (shimada et al. 2004) . this is concerning the fgf23 physiological function of regulating phosphate metabolism; on the other hand, mutations in fgf23 are implicated in a wide range of disorders. both autosomal dominant hypophosphatemic rickets and x-linked hypophosphatemic rickets were caused by excess fgf23, leading to hypophosphatemia (2000; jonsson et al. 2003; yamazaki et al. 2002) . fgf23 levels are increased tenfold above controls in patients with tumorinduced osteomalacia, a tumor associated syndrome of renal phosphate wasting (jonsson et al. 2003; yamazaki et al. 2002) . circulating fgf23 levels are also increased and correlate with disease burden in patients with fibrous dysplasia, a disorder in which normal bone is replaced by fibroosseous tissue (riminucci et al. 2003) . reduced fgf23 signaling also causes pathology. familial tumoral calcinosis is a disorder marked by hyperphosphatemia in which individuals develop calcified masses, often within the joints (lyles et al. 1985 ). in addition, in patients with chronic kidney disease (ckd), fgf23 concentrations are elevated constitutively and increase progressively as kidney function worsens (gutierrez et al. 2005) , suggesting a direct link between increased fgf23 and the progression of ckd (fliser et al. 2007) . it is a given that fgf23 has an important role in the physiological regulation of phosphate metabolism and its pharmacological significance in the pathogenesis of human disease. in the next step, it is necessary to prepare sufficient hfgf23 with activity for further mechanisms and pharmacological research. yamashita et al (2000) has expressed fgf23 in cho mammalian cell. it is well known that the mammalian cell expression system is more complicated and costly from the standpoint of culture requirements, making this strategy difficult to use in the development of largescale fermentation processes. furthermore, the preparation of sufficient amounts of virus for large-scale expression is time consuming. to overcome this shortcoming, plotnikov et al. (2000) expressed fgf23 in escherichia coli in the form of an inclusion body; however, because the inclusion bodies must be denatured and annealed, it was difficult to produce bioactive protein. our previous experiments showed that fgf23 without using this fusion strategy was least effective in expression and solubility properties. in recent years, small ubiquitin-related modifier (sumo) has become an effective biotechnological tool as a fusion system to enhance soluble expression of proteins and decrease proteolytic degradation, which could not be achieved by traditional expression systems sun et al. 2008) ; then, sumo is post-translationally and enzymatically cleaved from the desired protein by sumo c-terminal hydrolases-isopeptidases . various proteins, such as sars virus protein (zuo et al. 2005) , mmp13 (marblestone et al. 2006) , egf (sun et al. 2008) , metallothionein ), kgf2 (wu et al. 2009 ), and fgf21 (wang et al. 2010) , have been expressed successfully and purified using this fusion strategy. thus, we also cloned a sumo fragment and constructed an expression plasmid-containing sumo and human fgf23. the results show that this novel method of protein expression can promote significantly greater rfgf23 levels, facilitating the dissolution of rfgf23 in the soluble fraction for purification and producing native n-terminal recombinant protein with its bioactivity preserved. primers, plasmids, and strains all primers were synthesized chemically by beijing sunbiotech co. ltd (china) . the puc vector containing human fgf23 cdna sequence (aa25-251, genbank accession number bc069333 for expression of the protein without the first 24 amino acids) was purchased from proteintech group, inc. all plasmids and the strains are obtained from the laboratory (engineering research center of bioreactor and pharmaceutical development, ministry of education, jilin agricultural university china): the e. coli strain dh5α was used as the host for all cloning experiments, while protein expression studies were performed using e. coli strain rosetta (de3) as host; the vector of pet3c, pet22b and the expression strain of bl21(de3) were used for optimal expression and pet3c-sumo was used as the template of the sumo gene. the sumo protease was provided by dr. yadong huang from jinan university. the strategy for the construction of four kinds of rfgf23 recombinant plasmid is illustrated in fig. 1 . first, pcr was applied to amplify the dna fragments encoding the core fragment of rfgf23 with the forward fl and reverse r23 primers, including bamh i sites, using the puc vector according to the human fgf23 mrna coding sequence and the sumo fragment c-terminal sequence, five primers were designed and synthesized. as shown in fig. 1 , sumo-fgf23 and fgf23 were synthesized by pcr. the detailed steps are provided in the "material and methods" containing a human fgf23 coding sequence as the template. then, the core fgf23 fragment was prepared for linking to sumo; on the other hand, the fgf23 fulllength gene without fusion of sumo was created according to the procedure described above, except that the forward primer of f included the nde i sites. second, the sumo linker was generated using pet3c-sumo (plasmid-containing sumo fragment) as the template, p including nde i sites and the sumo linker (rl) as the forward and reverse primers, respectively. then, using the sumo linker and the core fgf23 fragment as the common template, p including nde i sites and r23 primers including bamh i sites as the forward and reverse primers, respectively, the sumo-fgf23 fusion gene construct was created using the same procedure described above. third, the fgf23 full-length gene and the sumo-fgf23 fusion gene were digested with ned i and bamh i, respectively, and then ligated into previously digested pet-3c, pet22b to create four kinds of new expression vectors, i.e., pet3c-fgf23, pet3c-sumo-fgf23, pet22b-fgf23, and pet22b-sumo-fgf23. correct insertion of sequences was confirmed by dna sequencing of each plasmid. the four kinds of fgf23 expression vector constructed above were transformed into competent cells of strains rosetta(de3) and bl21(de3). the transformants were grown in 4-ml luria-bertani (lb) medium containing 100 μg/ml ampicillin and 1% glucose at 37°c. when od600 reached 0.6, isopropyl-thio-β-galactopyranoside (iptg) was added to a final concentration of 1 mm. the culture was incubated at 37°c for 4 h with shaking at 220 rpm. the expression of each culture was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and the expression level of rhfgf23 was determined by densitometer scanning. the colony with the highest expression level was used in the subsequent experiments. an analysis was performed to determine the effects of the three factors (iptg concentration, temperature, and time after induction) on the expression yield and productivity of soluble fgf23 in rosetta(de3)/pet22b-sumo-fgf23. the recombinant bacteria were harvested by centrifugation at 30,000 × g for 5 min at 4°c. cell pellets were suspended in tris-hcl buffer at a concentration of 20 mm and dissolved by sonication. the suspensions were centrifuged at 30,000 × g for 30 min at 4°c. the clear supernatant (soluble fraction) was collected, and the remaining pellets (insoluble fraction) containing inclusion bodies were resuspended in an equal volume of lysis buffer. both soluble and insoluble fractions were analyzed by 15% sds-page. an overnight culture of e. coli strain rosetta(de3) transformed with the expression vector pet22b containing the sumo-fgf23 gene was diluted 1:50 into 2 l of growth medium (lb medium containing 100 μg/ml of ampicillin). the culture was incubated at 37°c until its optical density at 600 nm was 0.6. protein expression was induced by the addition of 0.4 mm iptg at 16°c. after 19 h of additional incubation, the cells were harvested, resuspended in 20 mm tris-hcl buffer (ph08.0) containing 2 m urea, and sonicated. the resulting cell lysate was cleared of bacterial debris by ultracentrifugation, and the fusion protein with his 6 -tagged n-terminal sumo was purified by deae sepharose ff column chromatography. sumo-fgf23 flows through the deae column with binding buffer (20 mm tris-hcl+2 m urea, ph08.0), followed by further purification with nickel nitrilotriacetic acid resin (ni-nta, invitrogen). the ni-nta resin was washed with binding buffer (20 mm tris-hcl+2 m urea, ph08.0) until od280 of the effluent reached baseline conditions. contaminating proteins were eluted from the column with wash buffer (20 mm tris-hcl+2 m urea containing 25 mm imidazole, ph08.0). finally, his 6 -tagged sumo-fgf23 protein was collected from the column with elution buffer (20 mm tris-hcl+2 m urea containing 250 mm imidazole, ph08.0). samples taken at the elution peak were pooled. the purity of sumo-fgf23 was assessed using sds-page, and the concentration was evaluated by the bradford method. to remove imidazole and urea, the fusion protein obtained from the affinity column was subjected to a buffer change to tbs using dialysis in 20 mm tris buffer (ph08.0) through a semipermeable membrane (cutoff, 10,000 da). the resulting protein solution was treated with sumo protease to release the recombinant fgf23. in a standard cleavage reaction, 50 μg of fusion protein (37.6 kda) in a 50-μl digestion buffer (50 mm tris-hcl, ph08.0, 0.15 m nacl, 1 mm dtt) was incubated overnight with five units of sumo protease (provided by dr. yadong huang from jinan university) at 4°c. the result of the cleavage reaction was monitored by sds-page. the target protein could be visualized by conventional sds-page. purification and the authenticity assay for rfgf23 after sumo protease cleavage, the reaction mixture contained his 6 -tagged n-terminal sumo, fgf23 target protein, sumo-fgf23 fusion protein, and sumo protease. then, the reaction mixture was passed through a miniature ni-imac column. the sumo, sumo-fgf23, and sumo protease stayed bound to the ni-nta resin, and only rfgf23 flowed through the column with the digestion buffer; these products were desalted with a sepharose g50 column. the purified rfgf23 was immunoblotted with a polyclonal mouse anti-fgf23 antibody (santa cruz biotechnology, inc.,) according to the manufacturer's protocol. immunoreactive bands were visualized using a dab kit (boshide, inc., wuhan, china). furthermore, the target protein were identified by matrix-assisted laser desorption/ionization tandem time-of-flight (maldi-tof/tof) mass spectrometry analysis (technical services provided by the shanghai applied protein technology co. ltd.). for high performance liquid chromatography (hplc) analysis, the purified rfgf23 was desalted and then loaded onto a c18 column. the elution was conducted using a linear gradient of 30-70% acetonitrile at a flow rate of 0.8 ml/min in the presence of 0.1% trifluoroacetic acid. the fractions containing rfgf23 were pooled. the concentration of rfgf23 was evaluated by the bradford method. in vitro activity assay of rfgf23 to assess the biological activity of rfgf23 proteins at the cellular level, we studied the ability of the protein to activate 44/42 map kinase in the glioma u251 cell line, which expresses abundantly fgf23 cognate receptors fgfr1, compared to commercial fgf23, which is also expressed in e. coli. the glioma u251 cells (1,000,000 cells/dish) were serum starved for 16 h and then stimulated with rfgf23 (100 ng/ ml), commercial fgf23 (100 ng/ml), or pbs for 10 min. after stimulation, the cells were lysed, and cellular proteins were resolved on sds-polyacrylamide gels and transferred electrophoretically to nitro cellulose membranes; then, the protein blots were probed with antibodies to phosphorylated 44/42 map kinase and non-phosphorylated 44/42 map kinase according to the manufacturer's protocol. the protein levels of p-erk, erk, were examined by western blot with actin as a loading control. all antibodies were from santa cruz biotechnology. in vivo activity assay of rfgf23 all described procedures that involved animals and their care were approved by the institutional animal care and use committee of jilin university and were performed in accordance with institutional guidelines for animal experiments. wistar adult male rats (weighting 220-250 g) were divided randomly into three groups: the treatment group (rfgf23, n010), control group (commercial fgf23, n0 10), and the negative control group (nc, saline n010). the phosphaturic activity of rfgf23 was examined in normal rats by a published protocol (goetz et al. 2007 ). briefly, the animals were fed with a complete, fixed-formula diet containing 0.94% phosphate for 2 weeks, and, then, they fasted overnight for 8-12 h. three hours before the injection, 0.5 ml of blood samples were collected from the tail. after the i.v. administering 0.1 μg/kg of body weight of either rfgf23, commercial fgf23, or saline, we took blood samples at 1, 2, and 3 h. phosphate concentrations in the serum were determined using a phosphomolybdic acid detection kit (nianjing jiangcheng co.). values are expressed as mean ± standard error of mean. comparisons of mean values between two time points were performed using the student's t test. significant differences were considered at p<0.05. cell assay were performed in triplicate. all experiments were repeated at least three times. to synthesize the full-length fgf23 and sumo-fgf23 fusion gene composed of sumo and the core fgf23 fragment, five special primers were designed (table 1 ). the synthesis strategy is described in fig. 1 , and the detailed procedure is included in the "materials and methods" section. the pcr products of sumo, fgf23, and sumo-fgf23 are shown in fig. 2 . the final pcr products (the full-length fgf23 and sumo-fgf23) were digested with two restriction enzymes (ned i and bamh i) and cloned into the expression vectors pet-3c and pet22b, respectively. the sequence of the target gene was confirmed by automated dna sequencing. the results of pcr products showed that the sequences of human to achieve greater productivity of rfgf23, we performed several small-scale expression studies. in the experiments, we used host bacteria rosetta(de3) and bl21(de3) and four kinds of recombinant plasmid for expression screening. the results showed that, except for the combination of pet22b-sumo-fgf23 and pet3c-sumo-fgf23, the two kinds of plasmid, and the rosetta(de3) host strain (fig. 3a) , all other combinations had no target protein expressed. we chose the rosetta(de3)/pet22b-sumo-fgf23 for optimization of induction conditions for soluble rfgf23. recombinants were inoculated in fresh lb medium and incubated in a shaking incubator at 37°c until the od600 was 1.0 to 1.2. then, iptg was added to a final concentration of 0.4 mm for 19 h at 16°c for the induction of expression. recombinant bacterial cells were collected by centrifugation and lysed by sonication. the supernatants and pellets were collected and analyzed using 15% sds-page. the results showed that the molecular weight of the expression product was 37 kda, which corresponds to the predicted size of sumo-fgf23. the target protein is more than 35% of the total cellular protein, and the soluble fraction was as much as 80% of the total expressed recombinant proteins (fig. 3b) . concerning optimal cell growth conditions for protein production, we screened for suitable iptg concentration, temperature, and time after induction for the expression yield and productivity of soluble fgf23 in rosetta(de3)/ pet22b/sumo-fgf23. we found that lower temperature (16°c), lower iptg concentration (0.4 mm), and longer induction (19 h) can express fgf23 protein significantly in soluble form, while most of the sumo-fgf23 protein was fig. 2 synthesis of fgf23 and sumo-fgf23 by pcr. the strategy for synthesizing fgf23 and sumo-fgf23 was described in the "material and methods." the molecular weight of the pcr fragment containing sumo and fgf23 was shown in a. lane 1 sumo fragment (340 bp); lane 2 fgf23 fragment (705 bp). the pcr product of the sumo-fgf23 fusion gene was shown in b. it was 1,018 bp in length the results showed that, the rosetta(de3) host strain, which were transformed by either pet22b-sumo-fgf23 or pet3c-sumo-fgf23, were effective for target protein expressed, and the fusion protein molecular mass was approximately 37.6 kda, corresponding to the predicted molecular weight. a m protein molecular weight marker (in kilodalton, takara); lanes 1-2 uninduced and induced rosetta(de3)/pet22b, respectively, as control; lanes 3 and 4 uninduced and induced rosetta (de3)/pet22b-sumo-fgf23, respectively; lane 5 induced rosetta (de3)/pet3c-sumo-fgf23. b lanes 1-2 the precipitation and supernatant of rosetta(de3)/pet22b-sumo-fgf23 were induced by 0.4 mm iptg for 19 h at 16°c; m protein molecular weight marker (in kilodalton, fermentas). analysis of the supernatant using 15% sds-page showed that the soluble sumo-fgf23 was expressed 80% at 16°c for 19 h in total supernatant protein expressed in the inclusion body at 37°c and 25°c in our previous experiment (data not shown). according to the isoelectric point of fusion protein, deae sepharose ff was chosen for the purification of sumo-fgf23. small amounts of host proteins were removed from sumo-fgf23 after it was purified with a deae sepharose ff column, except for most of the host dna (esm fig. s1) . a ni-nta affinity column was chosen for further purification. proteins without 6× his tags were removed from the ni-nta resin using washing buffer containing 25 mm imidazole; sumo-fgf23 was eluted using elution buffer containing 250 mm imidazole. sds-page analysis of samples taken from this step showed that the purity of sumo-fgf23 reached 94% (fig. 4a) . a sumo protease recognition sequence immediately upstream of the target peptide allowed the latter to be released from the fusion with sumo without leaving any unwanted residues upon treatment with sumo protease. to achieve maximal release of fgf23 protein, the cleavage reaction was performed overnight at 4°c. the efficiency of the cleavage was monitored by sds-page, and the slow disappearance of the band of the fusion protein at 37 kda (fig. 4b) was observed. isolation of recombinant rfgf23 from the cleavage mixture and further characterization of rfgf23 by western blot and hplc when the target protein was fused directly to the c-terminus of sumo, cleavage by sumo protease 1 resulted in the release of the target protein with the desired n-terminal amino acid sequence. in our studies, after the purified fusion protein was diluted and cleaved by sumo protease 1, the cleavage mixture was purified by ni-nta resin. sumo, sumo protease 1, and sumo-fgf23 fusion protein containing his tags that were affiliated with ni-nta resin, but only rfgf23, flow through the column with the digestion buffer. our results showed that rfgf23 was highly purified by sds-page (fig. 5a ) and could react with the human fgf23 polyclonal antibody by western blot (fig. 5b) . hplc analysis of the target protein showed a major peak of rfgf23, with a retention time of 14.770 min; the purity exceeded 90% (fig. 5c) . also fgf23 protein was successfully identified by maldi-tof/tof. details of the protein was listed in esm fig. s2 . to test the activity assay of the rfgf23, we stimulated the glioma u251 cells with both rfgf23 and commercial fgf23 and then examined whether rfgf23 protein can induce the phosphorylation of 44/42 mitogen-activated protein (map) kinase (p44/42 mapk) in the glioma u251 cells. as illustrated in fig. 6a , the phosphorylation of mapk were activated by either rfgf23 or commercial fgf23; also, the rfgf23-induced phosphorylation of erk (141% activation, compared with negative-treated group) was more than that of commercial fgf23 (116% activation, compared with negative-treated group). the results suggested that the rfgf23 produced by sumo fusion method had better biological activity than the commercial fgf23. here, we speculate that the commercial fgf23 may be fig. 4 sds-page analysis of purification and cleavage of sumo-fgf23. after recombinant bacteria of rosetta(de3)/pet22b-sumo-fgf23 were treated by sonication and centrifugation, the supernatants were loaded on a deae sepharose ff and ni-nta orderly. the purification results for sumo-fgf23 are shown in the a. a m protein molecular weight standard (in kilodalton, fermentas); purified sumo-fgf23 eluted from ni-nta column; the purified sumo-fgf23 was digested by sumo protease at 4°c overnight. as seen in b, rfgf23 was cut from the sumo-fgf23 by sumo protease. b m protein molecular weight standard (in kilodalton, takara); recombinant his 6tagged sumo-fgf23 fusion protein, rfgf23, and his 6 -tagged su-mo as cleavage product, respectively expressed in the inclusion body in e. coli or in some other form that was not fused with sumo. to evaluate further the biological activity of rfgf23, we constructed the rat models as described in the "materials and methods" section. according the reference of goetz (shimada et al., 2004) , mice were administered with either rfgf23 or commercial fgf23 at 0.1 μg/kg (i.v.) in the experiments. blood samples were collected 3 h before and 1, 2, and 3 h after injection. the plasma levels of phosphate were determined by a phosphomolybdic acid detection kit. the statistical results are described in the column graph shown in fig. 6b . we can see that comparison of the phosphate levels before and after treatment disclosed that both rfgf23 and commercial fgf23 had hypophosphatemic effects over 1-3 h. compared to commercial fgf23, however, rfgf23 afforded a better hypophosphatemic effect (fig. 6b) . the better hypophosphatemic effect of rfgf23 than commercial fgf23 was considered due to the increased bioactivity of rfgf23 produced by sumo fusion method. the discovery of fgf23 not only revealed that there is a regulatory system of serum phosphate but also expanded the spectrum of endocrine diseases. it is necessary to develop methods for abundant production of fgf23 with specific bioactivity for specific treatment of fgf23-related diseases in the future; therefore, we have reported a novel strategy to express and purify recombinant hfgf23 greatly. in our experiment, we have proved that fgf23 is least effective in expression properties as an unfused protein, regardless of either of the two host strain (data were not shown). whereas, fgf23, when fused with sumo, showed higher amount of expression, but only in the host strain of rosetta(de3) not bl21(de3). because on the one hand, sumo have a chaperone-like activity, the translational fusion of sumo to other proteins often results in significantly improving expression of those proteins in e. coli malakhov et al. 2004; marblestone et al. 2006; panavas et al. 2009; zuo et al., 2005) ; on the other hand, rosetta(de3) strain can provide additional trnas for e. coli rare codons (aua, agg, aga, cua, ccc, gga), thereby increasing fgf23 gene expression. taken together, for protein efficient expression, both were indispensable. our data also showed that soluble sumo-fgf23 concentration was over 25% of all protein at optimal expression conditions (induced by 0.4 mm iptg for 19 h at 16°c), compared to that when induced by 1 mm iptg at 37°c or 25°c, most of sumo-fgf23 protein was expressed in inclusion body (data were not shown). in fact lower c hplc analysis of the purified rfgf23. the purity of rhfgf23 was further evaluated by hplc analysis using a c18 column. as seen from the chromatogram, the y-axis indicates the absorbance, while the x-axis represents elution time (in minutes). the main peak eluted at 14.770 min. the purity of purified rhfgf23 was greater than 90% temperature and lower iptg concentration indeed make protein fold significantly decelerated and increase soluble protein level. in addition, a long induction period is also necessary. during the purification, in the presence of 2 m urea denaturants, both sumo-fgf23 solution and its affinity with ni-nta were significantly altered, whereas, in the absence of 2 m urea, sumo-fgf23 is not well soluble in solution, but also all flew through nickel column (our previous experiment have proved this point, data were not shown). we speculate that, most likely, fgf23 with 227 amino acids covered the six histidine sites before the sumo tag with 108 amino acids. adding 2 m urea into the buffer made the six histidine sites before the sumo tag exposed, thereby increasing the affinity for ni-nta resin. because fgf23 is a systemic humoral factor, its renotropic function indicates that the kidney may bear a unique receptor specific for fgf23. despite our previous attempts with the use of human kidney cell lines, phosphorylation responses were not well observed after rfgf23 treatment. we tried the glioma u251 cells for overexpressing fgfr1 to evaluate rfgf23 biological activity, and a better response is obtained (seen fig 6a) although in the case of the absence of a klotho receptor. recent etiological studies indicate that high fgf23 levels are associated with increased mortality and cardiovascular dysfunction, both in patients with ckd and also in subjects with preserved renal function (juppner et al. 2010; shimada et al. 2004) . whether fgf23 has a direct action on cardiovascular tissues because klotho is also not expressed there, the study of cell assay strategy is a good answer to the hypothesis put forward. many experiments have documented that fgf23 reduces serum phosphate mainly by the two mechanisms of both inhibiting proximal tubular phosphate reabsorption and intestinal phosphate absorption. specific explanation is that fgf23 suppresses the expression of type 2a and 2c sodiumphosphate cotransporters which mediate the physiological phosphate reabsorption in proximal tubules (shimada et al. 2004) ; the other is that fgf23 decreases the expression of 25-hydroxyvitamin d-1α-hydroxylase and at the same time enhances 25-hydroxyvtamin d-24-hydroyxlase expression (shimada et al. 2004) . by modifying the expression of these vitamin d metabolizing enzymes, fgf23 acts to reduce serum 1,25-dihydroxyvitamin d [1,25(oh)2d], which stimulates intestinal calcium and phosphate absorption. in our studies, we can also see that the changes in plasma phosphate levels all showed significant differences after either injecting rfgf23 or commercial fgf23, compared to fig. 6 in vitro and in vivo activity assay of rfgf23. a in vitro activity assay of rfgf23. the glioma u251 cell lines were treated with either rfgf23 or commercial fgf23 for 10 min. the protein levels of phosphorylation of 44/42 map kinase was examined by western blot with actin as a loading control (values on the top of western blots represent the mean optical density ratio in three independent experiments, vs. negative control group). b in vivo activity assay of rfgf23. after either rfgf23 or commercial fgf23 (0.1 μg/kg, i.v.) was administrated to wistar adult male rats, blood samples were collected at 1, 2, and 3 h. plasma phosphate concentrations were measured as described in "materials and methods." data point represent the mean±sd (n010). (*p< 0.05, **p<0.01, vs. 3 h before the rfgf23 injection; #p<0.05 vs. 3 h before the commercial fgf23 injection; bt before treatment; at after treatment) negative control, but administration of rfgf23 resulted in a time-dependent better reduction in circulating levels of phosphate than commercial fgf23. we speculated that different effects might be due to different expression form in e. coli. in summary, rfgf23 was successfully expressed in e. coli rosetta(de3) from sumo-fgf23 fusion gene. the resulting fusion protein could be cleaved by sumo protease to give free rfgf23 and then isolated by the ni-nta affinity column. the identity of the purified protein was confirmed by western blot, and its purity was determined by hplc analysis. the recombinant fgf23 can induce phosphorylation of 44/42 map kinase (p44/42 mapk) in the glioma u251 cell line. the results of animal experiments in vivo also showed that rfgf23 produced by using this method can decrease the concentration of plasma phosphate in normal rats fed with a fixed formula diet. this study demonstrated that sumo, when fused with fgf23, was able to promote the soluble expression of the latter in e. coli, making it convenient to purify rfgf23 with its bioactivity preserved. sumo fusion technology for difficult-to-express proteins fibroblast growth factor 23 (fgf23) predicts progression of chronic kidney disease: the mild to moderate kidney disease (mmkd) study molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members fibroblast growth factor-23 mitigates hyperphosphatemia but accentuates calcitriol deficiency in chronic kidney disease expression and purification of glutathione transferase-small ubiquitin-related modifiermetallothionein fusion protein and its neuronal and hepatic protection against d-galactose-induced oxidative damage in mouse model fibroblast growth factor 23 in oncogenic osteomalacia and xlinked hypophosphatemia fgf-23: more than a regulator of renal phospate handing? genetic transmission of tumoral calcinosis: autosomal dominant with variable clinical expressivity sumo fusions and sumo-specific protease for efficient expression and purification of proteins comparison of sumo fusion technology with traditional gene fusion systems: enhanced expression and solubility with sumo sumo fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems crystal structures of two fgf-fgfr complexes reveal the determinants of ligand-receptor specificity fgf-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting fgf-23 is a potent regulator of vitamin d metabolism and phosphate homeostasis expression and purification of human urodilatin by small ubiquitin-related modifier fusion in escherichia coli high-level expression and purification of soluble recombinant fgf21 protein by sumo fusion in escherichia coli expression and purification of human keratinocyte growth factor 2 by fusion with sumo identification of a novel fibroblast growth factor, fgf-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain increased circulatory level of biologically active fulllength fgf-23 in patients with hypophosphatemic rickets/osteomalacia expression and purification of sars coronavirus proteins using sumo-fusions acknowledgements we wish to thank ms peng jing for western blot analysis. the project was supported in part by research grants from project of wenzhou sci & tech bureau (y20100181) and the national high technology research and development program (2011aa02a113). key: cord-321602-88b2h06y authors: lv, chenfei; shi, tingting; zhu, pengpeng; peng, xing; cao, shangshang; yan, yan; ojha, nishant kumar; liao, min; zhou, jiyong title: construction of an infectious bronchitis virus vaccine strain carrying chimeric s1 gene of a virulent isolate and its pathogenicity analysis date: 2020-08-19 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10834-2 sha: doc_id: 321602 cord_uid: 88b2h06y abstract: infectious bronchitis virus (ibv) is a member of genus gamma-coronavirus in the family coronaviridae, causing serious economic losses to the poultry industry. reverse genetics is a common technique to study the biological characteristics of viruses. so far, there is no bac reverse genetic system available for rescue of ibv infectious clone. in the present study, a new strategy for the construction of ibv infectious cdna clone was established. the full-length genomic cdna of ibv vaccine strain h120 was constructed in pbac vector from four ibv fragment subcloning vectors by homologous recombination, which contained the cmv promoter at the 5′ end and the hepatitis d virus ribozyme (hdvr) sequence and bovine growth hormone polyadenylation (bgh) sequence after the polya tail at the 3′ end of the full-length cdna. subsequently, using the same technique, another plasmid pbac-h120/scs1 was also constructed, in which s1 gene from ibv h120 strain was replaced with that of a virulent sc021202 strain. recombinant virus rh120 and rh120/scs1 were rescued by transfecting the plasmids into bhk cells and passaged in embryonated chicken eggs. finally, the pathogenicity of both the recombinant virus strains rh120 and rh120/scs1 was evaluated in spf chickens. the results showed that the chimeric rh120/scs1 strain was not pathogenic compared with the wild-type ibv sc021202 strain and the chickens inoculated with rh120/scs1 could resist challenge infection by ibv sc021202. taken together, our results indicate that bac reverse genetic system could be used to rescue ibv in vitro and ibv s1 protein alone might not be the key factor for ibv pathogenicity. key points: • bac vector was used to construct ibv full-length cdna by homologous recombination. • based on four subcloning vectors, a recombinant chimeric ibv h120/scs1 was constructed and rescued. • pathogenicity of h120/scs1 was similar to that of h120, but different to that of sc021202. infectious bronchitis virus (ibv) is a member of the coronaviridae family in the order nidovirales. it is a highly pathogenic virus that causes serious economic losses to the world poultry industry (cavanagh 2005; cook et al. 2012) . ibv is an enveloped virus having a single-stranded, positivesense rna genome of 27.6 kb in size (boursnell et al. 1987) . because of the high degree of genome sequence variation, ibv has many genotypes circulating at any time in poultry farms worldwide (jackwood 2012) , making it difficult to control or prevent its spread through vaccination. reverse genetics is a common in vitro method to study virus biological characteristics. the development of this technique allowed researchers to manipulate the ibv genome and rescue of virus in vitro (shan et al. 2018) . previously, the most common technique to obtain chenfei lv and tingting shi contributed equally to this work. the full-length cdna of ibv was the cdna ligation (youn et al. 2005; zhou et al. 2013) . by using this method, small pieces of ibv genomic cdnas were amplified, digested with restriction enzymes (re), and then ligated. the full-length cdna with t7 promoter attached to 5′ end of the ibv genome was transcribed and transfected into bhk cells by electroporation to rescue the virus (casais et al. 2001) . another reverse genetic system for ibv was based on targeted rna recombination. this method enabled the modification of the ibv genome at its 3′ terminal by targeted rna recombination (van beurden et al. 2017) . by using this method, a recombinant h52 strain carrying the spike glycoprotein ectodomain of mhv was constructed, which had the capability to grow in a mammalian cell lines (van beurden et al. 2017 ). however, targeted rna recombination is only useful for the modification of 3′-end of the ibv genome and is difficult to modify the 5′-end polymerase gene, because the construction of donor rna vectors entering this region is hindered (van beurden et al. 2017) . bacterial artificial chromosomes (bac) vector reverse genetic system is also a common method to rescue coronaviruses in vitro. the first coronavirus full-length genomic cdna in bac was constructed for tgev (almazan et al. 2000) . later on, several coronavirus, such as sars-cov (almazan et al. 2006) , fipv (balint et al. 2012) , and mers-cov (scobey et al. 2013) , were rescued successfully using that technique. however, there is no bac reverse genetic system currently available for the rescue of ibv. the spike protein of ibv is posttranslationally cleaved into two subunits, s1 and s2, where s2 is anchored into the viral envelop and is important for the membrane fusion. s1 comprises the head domain of spike and is responsible for host receptor binding (wickramasinghe et al. 2011) . the s1 subunit is the important immunogenic component that contains epitopes for neutralizing antibody (ignjatovic and sapats 2005) . in this study, a new strategy for construction of ibv infectious clone was established. a nephropathogenic strain sc021202 (close to ibv gi-22 genotype), which causes severe kidney damage and mortality to infected chickens (zhou et al. 2004 ) was selected as donor of ibv s1 gene. first, infectious clone of ibv vaccine strain h120 (rh120) was rescued using the bac reverse genetic system. then, a chimeric ibv virus rh120/scs1 with the h120 backbone replacing its s1 subunit with that of virulent sc021202 isolate was constructed. lastly, the rh120/scs1 was used to inoculate specific pathogen free (spf) chickens for the preliminary study of the function of s1 of ibv field isolate on virus pathogenicity and the potential of recombinant virus as a vaccine candidate was also evaluated. the strains of ibv sc021202 (genbank no.: eu714029.1) and h120 (genbank no.: fj888351.1) were stored in our laboratory. viruses were propagated and titrated in the allantoic cavity of 10-day-old embryonated spf chicken eggs. baby hamster kidney (bhk-21) cell line was stored in our laboratory and cultured at 37°c in dulbecco's modified eagle's medium (dmem; gibco, carlsbad, ca) supplemented with 10% fetal bovine serum (fbs, biological industries, israel), penicillin (100 u/ml), and streptomycin (100 μg/ml). primary chicken embryonated egg kidney (cek) cells were prepared from 17-19-day-old spf chicken embryonated eggs and maintained in dmem supplemented with 10% fbs at 37°c in 5% co 2 atmosphere. competent cells were prepared according to the instructions provided by ultra-competent cell preps kit (sang biotech, china). pbelobac11 (called pbac in the subsequent texts) vector was a gift from professor yaowei huang of zhejiang university. construction scheme of a full-length cdna clone of ibv h120 to construct the full-length cdna of ibv h120 strain, total rna was extracted from the allantoic fluid of spf chicken embryonated eggs infected with h120 and transcribed to cdna by reverse transcriptase using the thermo scientific revertaid first-strand cdna synthesis kit (thermo fisher, usa). four cdna fragments (f1, f2, f3, and f4) covering the whole h120 genome sequence were amplified separately from the cdna template with the primer sets (f1-f and f1-r, f2-f and f2-r, f3-f and f3-r, f4-f and f4-r) shown in table 1 , which also contained sequences for homologous recombination. the procedure for the construction of full-length cdna of ibv h120 has been shown in fig. 1a . the amplified 4 fragments (f1, f2, f3, and f4) carrying the same homologous recombination sequences as the pbac vector were cloned into modified linearized pbac vector. homologous recombination reaction was carried out according to the instructions provided with clonexpress one-step cloning kit (vazyme biotechnology, china). the resulting plasmids were named as pbac-f1, pbac-f2, pbac-f3, and pbac-f4. bamhi enzyme site in the first fragment was mutated as a marker (a5472c) for identification of rescued virion through amplification of the f1 fragment with primers f1a5472c-f and f1a5472c-r containing mutation site (table 1) . for the 5′utr and 3′utr fragments, the primers (5′utr-f and 5′utr-r, 3′utr-f and 3′utr-r) containing the (table 1) , and the nucleotide sequences were separately amplified. the first and fourth subcloning vectors pbac-f1 and pbac-f4 were digested with bamhi and xhoi restriction enzymes (takara, japan), respectively, resulting in a linearization of the vectors (fig. 1a) . the 5′utr was fused to the first subcloning vector construct, and the 3′utr was fused to the fourth construct by homologous recombination to obtain the first subcloning pbac-f1 and fourth subcloning pbac-f4. the first subcloning vector construct was modified by adding the cmv (cytomegalovirus) promoter sequence in the 5′ terminal of the first fragment, and then hdvr (hepatitis delta virus ribozyme) sequence and bgh (bovine growth hormone polyadenylation signal) sequence were added to the 3′ terminal of the fourth fragment after the poly(a) by homologous recombination with the primers (cmv-f and cmv-r, hb-f and hb-r) listed in table 1 . subsequently, the f2 and f4 fragments were amplified with the primer sets (f2-f1)-f and (f2-f1)-r and primer sets (f4-f3)-f and (f4-f3)-r (table 1) , respectively. they were fused to the linearized plasmids pbac-f1 and pbac-f3 digested by xhoi to obtain two semi-subclones pbac-f12 and pbac-f34, respectively (fig. 1a) . finally, two semisubclones were fused using the same method with the primer sets (f34-f12)-f and (f34-f12)-r (table 1) to complete the full-genome cdna construction of ibv h120 (fig. 1a ). replacement strategy for chimeric recombinant h120/scs1 and generation of full-length cdna procedure used to construct chimeric h120 recombinant strain has been shown in fig. 1b . the linearized plasmid dna pbac-f4(hb)-δs1 lacking the s1 gene was reverse amplified from the plasmid pbac-f4 with primer set h120-f4-△s1-f and h120-f4-△s1-r (table 1 ) as a linearized vector. the sc021202 s1 gene containing same homology arms to those of the h120f4 was amplified from total rna of allantoic fluid of spf chicken embryonated eggs infected with ibv sc021202 by rt-pcr with the primers scs1-f and scs1-r listed in table 1 and was then inserted into pbac-f4-△s1 by the same homologous recombination process using the clonexpress one-step cloning kit (vazyme biotechnology, china). then, the third segment of ibv genome and the pbac-f4/scs1 was fused to obtain two semisubclones. subsequently, both of these semi-subclones were fused to complete the construction of full-genome cdna of ibv h120/scs1. bhk-21 cells were cultured in 6-well plates. when the cells grew to 60% confluency, 4 μg (200 μl/well) of pbac-h120 or pbac-h120/scs1 was transfected to the cells using jetprime transfection reagent (polyplus, france) according to the manufacturer's instructions. transfected cells were incubated for 6 h at 37°c. after the incubation, the culture medium was replaced with dmem with 2% fbs and cells were incubated at 37°c for 48 h. after completion of the incubation period, culture medium and cells were harvested by repeated freeze-thaw (three times) and named as rh120 and rh120/scs1 p0 generation of the rescued virus. this p0 generation was inoculated into 10-day-old spf chicken embryos. after 48 h, the allantoic fluid of chicken embryo was collected and blindly passaged for 3 generations. eid 50 of passaged virus was determined by the method of reed and muench. the p0 generation rescue virus and allantoic fluid of infected chicken embryo were used for rna extraction. reverse transcription reaction was carried out using a reverse transcription kit (thermo fisher, usa) to obtain cdna. ibv h120 strain was used as template for designing primers (rm-f and rm-r, table 1 ) before and after the introduced rescue marker (rm) to amplify a 621 bp fragment spanning between 5211 and 5831 bp of the viral genome. the pcr products were purified and ligated into pmd-18t vector (takara, japan) and then transformed into competent cells, and positive clones were picked for sequencing to identify rescue marker sites. the rescue virus was further inoculated into the spf chicken embryonated eggs and observed for development of dwarf embryo lesion. to examine the stability of rescued viruses, s1 fragment of the virus of different passages was amplified from total rna extracted from allantoic fluid of infected embryonated eggs by rt-pcr using the primers h120sc-s-f and h120sc-s-r (table 1 ). the amplified fragments were then sequenced and analyzed. cek cells prepared from 18-day-old chicken embryos were infected with h120, rh120, and rh120/scs1. the medium was removed 48 hpi, and infected cells were fixed with methanol:acetone (1:1) at − 20°c. considering that membrane protein (m protein) is one of the abundant structural proteins in coronavirus (neuman et al. 2011) , in-house mouse monoclonal antibody 2b3 to ibv m protein (1:1000) was used as primary antibody in immunofluorescence assay, which were produced by our research group by the method described previously (hu et al. 2007 ). bound primary antibody was detected with fluorescein-labeled antibody to mouse the 10-day-old spf chicken embryonated eggs were inoculated with ibv h120, rh120, and rh120/scs1. after 48 h, allantoic fluids were collected and subjected to sds-page analysis. the separated proteins were then transferred to a nitrocellulose membrane at 400 ma for 30 min using 25 mm tris-192 mm glycine buffer (ph 8.3) containing 20% methanol. the membrane was blocked with 5% skim milk in pbs (20 mm tris-hcl, ph 7.5, 150 mm nacl) for 1 h and then washed three times with wash buffer . after that, the membrane was incubated with monoclonal antibody 2b3 against ibv m protein (dilution 1:1000) for 1 h. the membrane was again washed three times with wash buffer at room temperature. after that, bound primary antibody was detected with horseradish peroxidaseconjugated goat anti-mouse secondary antibody (dilution 1:5000) (sigma-aldrich, usa). signals were analyzed by enhanced chemiluminescence using the ami600 system (ge healthcare, usa). a total of fifty 7-day-old spf chickens were divided into five groups and housed in different negative pressure isolators. ten chickens in each group were inoculated with 0.3 ml of 10 4.5 eid 50 of ibv h120, rh120, rh120/scs1, and sc021202 via intranasal route. birds in the control group were inoculated with the same volume of sterilized pbs. after every 2 days, oropharyngeal and cloacal swabs were collected and subjected to rt-pcr to check the virus shedding status. the dead chickens were examined by necropsy, and the tissues displaying gross lesions were collected. meanwhile, one chicken in the groups without mortality was randomly selected for dissection and observation of gross lesions in tissues. the tissues of trachea and kidney were sent to wuhan servicebio technology co., ltd. (hangzhou, china) for processing hematoxylin and eosin (h&e) staining, and pathogenic lesion was observed under the microscope. a total of forty 7-day-old spf chicks were divided into four groups and 10 chickens in every group were immunized with 0.3 ml of 10 4.5 eid 50 of ibv h120, rh120, and rh120/scs1 by intranasal inoculation, and chickens in the control group were inoculated with the same volume of sterilized pbs. after 7 and 14 days post-immunization, serum was collected and the antibody against ibv was detected by elisa. then, the chickens were challenged with 0.3 ml of 10 5.85 eid 50 of ibv sc021202 strain. clinical signs and mortality of chickens after the challenge were recorded every day. elisa ibv n recombinant protein (1 μg/well) in 0.01 m pbs (ph 7.4) was coated on a 96-well microtiter plates (canada jet biochemicals int'l. inc.) at 4°c overnight, followed by blocking with 200 μl blocking buffer for 2 h at 37°c. the plate was washed three times with pbst and then incubated with 100 μl chicken serum samples diluted in blocking buffer (1:500) for 1 h at 37°c. after washing three times with pbst, plates were incubated with 100 μl hrp-conjugated goat antichicken igg (kpl, usa) in blocking buffer for 1 h at 37°c. after washing three times with pbst, the colorimetric reaction was developed after incubating the plates with 100 μl chromogenic substrate for 10 min at 37°c. color development was stopped with 50 μl 2 m h 2 so 4 , and an optical density at 450 nm (od 450 nm) was recorded using elx800 universal microplate reader (bio-tek instruments, inc., usa). the full-length cdna of ibv h120 strain was constructed according to the procedure shown in fig. 1a . four fragments (f1, f2, f3, and f4) covering the whole genome were amplified by rt-pcr with the total rna of ibv h120 as template (fig. 2a) . after the addition of cmv promoter to the 5′-terminal of f1 fragment and hb sequence to the 3′-terminal of f4 fragment, all four fragments were individually cloned into pbac vectors resulting into the generation of four fragment subcloning vectors. afterwards, those fragments were fused together by homologous recombination. finally, the fulllength cdna of ibv h120 in pbac vector (pbac-ibv-h120fl) was obtained and confirmed by re digestion, which showed that the full length of pbac-ibv-h120fl was digested into two major bands with expected molecular size of around 24,741 and 7507 bp by xhoi and bamhi (fig. 2b) . the full-length cdna was further confirmed by sequencing (data no shown). additionally, we made another full genome construct in which s1 gene of ibv h120 was replaced with that of strain sc020102 and termed as pbac-ibv-h120/scs1. strategy for the clone construction has been shown in fig. 1b . the recombinant plasmid pbac-ibv-h120/scs1 was also confirmed by re digestion with similar results to that of pbac-ibv-h120fl (fig. 2b) . the full-length cdna was also further confirmed by sequencing (data no shown). generation and characterization of rescued virus rh120 and rh120/scs1 pbac-ibv-h120 and pbac-ibv-h120/scs1 plasmids were first transfected into bhk-21 cells to obtain the p0 generation of rescued virus rh120 and rh120/scs1. this p0 virus was passaged in spf chicken embryonated eggs for three times. the p3 generation of the rescued viruses caused typical dwarf embryo lesions (data not shown), and ibv-specific sequences were also amplified using the primers rm-f and rm-r (table 1) with the cdna of the p3 generation of the rescued viruses (fig. 3a) . the mutant oligonucleotide site that served as rescue virus marker (ggatcc → ggctcc) was also confirmed to be in the correct position (data not shown). the s1 gene of rh120/scs1 was also amplified and sequenced, which was confirmed to be the s1 gene of sc021202 (data not shown), indicating that the s1 gene of h120 was successfully replaced with that of a heterogeneous ibv strain in a chimeric rescued virus. as results shown in fig. 3b and fig. 3c , ibv viral protein could be detected in rh120 and rh120/scs1 infected cek cells by western blot analysis and ifa. these results indicated that recombinant virus rh120 and rh120/ scs1 were successfully rescued. subsequently, the rescued rh120 and rh120/scs1 were continuously passaged in chicken embryonated eggs for 8 generations. s1 gene fragment of each passage was sequenced and analyzed. changes in the s1 gene sequence were not found in the rescued viruses, which indicated that the s1 genes of rh120 and rh120/scs1 were stable during the repeated passages. the mutant oligonucleotide site (ggatcc → ggctcc) which served as a rescue virus marker was also present in all generations (data not shown). pathogenicity and immunoprotection of chimeric vaccine strain rh120/scs1 to examine the pathogenicity of ibv h120 strain carrying s1 gene of virulent isolate, same doses of h120, rh120, rh120/ scs1, and sc0201202 were inoculated into the 7-day-old spf chickens. chickens inoculated with sterilized pbs were used as negative control. results of rt-pcr indicated that the chickens infected with h120, rh120, and rh120/scs1 showed persistent shedding of virus until 13 dpi (fig. 4a , table 2 ). clinical signs, such as respiratory distress, were only observed in sc021202 group at 3-7 dpi, and not in h120, rh120, and rh120/scs1 groups. fatality occurred only in the sc021202 infection group with mortality rate of 30% (fig. 4b) . kidneys of dead chickens showed pale discoloration and were swollen with white urate deposits, showing a typical "spotted kidney"-like lesion (fig. 4c) . mortality and severe gross pathology in kidneys were not observed in h120, rh120, and rh120/scs1 infection groups as well as the negative control group ( fig. 4b and 4c ). severe histopathological changes were observed only in the kidneys and tracheas of chickens infected with strain sc021202 (fig. 4d) . the kidney section from dead chickens in the sc021202 group showed enlarged renal tubules with epithelial cell exfoliation inside the tubular lumen, necrosis of some epithelial cells, and increased exudate with inflammatory cell infiltration (fig. 4d) . in the tracheal section from dead chicken in the sc021202 group, we observed that the epithelial cells of the mucous layer were exfoliated, degenerated, and necrosed (fig. 4d) . no obvious histopathological changes were observed in h120, rh120, and rh120/scs1 groups (fig. 4d) . in order to find out whether the chimeric virus could provide immunoprotection against the virulent ibv isolate, the chickens infected with h120, rh120, and rh120/scs1 14 dpi were challenged with ibv strain sc021202. the results showed that all the chickens survived except those in the control group (fig. 4e) . the antisera collected from h120, rh120, and rh120/scs1 immunized chickens at 7 and 14 dpi were analyzed by elisa. as result showed in fig. 4f , chickens immunized by h120, rh120, and rh120/scs1 could produce antibody against ibv at 7 and 14 dpi but not in pbs control group (fig. 4f ). compared with other avian respiratory viruses, such as avian influenza virus and newcastle disease virus, it is difficult to manipulate ibv genome by reverse genetics due to its largesized genomic rna (lai 2000) and also the availability of very few permissible cell lines for ibv infection (ferreira et al. 2003) . ligation of restriction enzyme (re)-digested fragments is a method to obtain the full-length cdna of ibv genome for rescue of ibv (casais et al. 2001 ). however, the procedure for ligation of large dna fragments to form the full-length genomic cdna is complicated, timeconsuming, and inefficient. on the other hand, the full length of ibv genome is long and selection of the suitable re for ligation of the full-length cdna of ibv genome is difficult. hence, there is a need to establish an efficient reverse genetic system to rescue ibv in vitro. bac reverse genetic system is a common in vitro method to rescue coronavirus, such as tgev (almazan et al. 2000) , sars-cov (almazan et al. 2006) , fipv (balint et al. 2012) , and mers-cov (scobey et al. 2013) . ligation of dna fragments by homologous recombination can be avoided to select many re enzyme sites. but, so far, no bac vector combined with homologous recombination has been used for the construction of ibv full-length cdna. in this study, four dna fragments of around 7000-8000 bp covering full-length genome of ibv were separately amplified and cloned into the low-copy vector pbelobac11 by homologous recombination. the necessary component sample type 1 dpi 3 dpi 5 dpi 7 dpi 9 dpi 11 dpi 13 dpi h120 oropharyngeal 4/10 7/10 9/10 6/7 6/7 6/7 5/7 cloacal 5/10 5/10 3/10 6/7 6/7 4/7 3/7 rh120 oropharyngeal 10/10 6/10 6/10 4/7 4/7 7/7 6/7 cloacal 3/10 2/10 3/10 3/7 5/7 5/7 4/7 rh120/scs1 oropharyngeal 8/10 9/10 10/10 4/7 4/7 7/7 5/7 cloacal 3/10 4/10 7/10 2/7 5/7 3/7 6/7 sc021202 oropharyngeal 4/10 10/10 a 10/10 a 7/7 a 7/7 5/7 6/7 cloacal 2/10 10/10 a 7/10 a 5/8 a 3/7 4/7 4/7 negative control oropharyngeal 0/10 0/10 0/10 0/7 0/7 0/7 0/7 cloacal 0/10 0/10 0/10 0/7 0/7 0/7 0/7 a obvious breathing distress was observed sequences of 5′-utr, 3′-utr, promoter sequence from cmv as well as hdvr and bgh were added to the 5′ terminal of the first fragment and the 3′ terminal of the last fragment by the same way. added hdvr at the end of ibv genome will increase the efficiency of rescue of ibv infectious clones by maintaining the integrity of the 3′ terminal of ibv genome. using the same strategy, two fragments, each were again fused together into the subcloning vector, and finally, both (f) detection of antibodies to ibv n protein of antisera collected one week and two weeks after immunization from ibv h120, rh120, and rh120/scs1 immunized chicken by elisa vectors each carrying two fragments were fused into a fulllength cloning vector. construction of subcloning vector by homologous recombination simplifies the operation and improves the efficiency of the ligation. in this reverse genetic system, site mutation, sequence deletion, and insertion can easily be performed based on certain sequence sites in the backbone of subcloning vector. with the reverse genetics established in this study, recombinant h120 (rh120) strain and its chimeric virus rh120/ scs1 carrying s1 gene of a virulent ibv sc021202 strain were rescued successfully. the pathogenicity of rh120/ scs1 was found to be similar to those of h120 and rh120 and was not able to cause pathological changes as shown in the case of strain sc021202-infected chickens. previous reports showed that s1 protein of ibv was responsible for pathogenicity, tropism, and antigenicity (hulswit et al. 2016; wickramasinghe et al. 2011) . however, our result indicates that only the s1 gene of sc021202 isolate is not a determinant of the pathogenicity of ibv since rh120/scs1 with h120 as the backbone and chimeric sc021202 s1 gene did not cause obvious sickness to the sensitive spf chickens as caused by the sc021202 strain. the results of this study are in agreement with previous studies showing that the s1 gene has little effect on pathogenicity (armesto et al. 2011; hodgson et al. 2004; jiang et al. 2020) . whether the s1 gene is the determinant of the pathogenicity of ibv might depend on individual isolate, which needs further study to confirm. finally, the immunization of h120, rh120, and rh120/ scs1 could protect the chicken against the challenge by ibv strain sc021202, which indicated that the recombinant clones constructed for ibv in this study may be useful for the development of novel ibv vaccine. in summary, in this study, with homologous recombination technology, an efficient reverse genetic system based on bac vectors for rescue of ibv infectious clone was established. this will provide a useful tool for further study of the pathogenicity-related genes of ibv and the development of vaccine against the virus. author contributions jyz, ml, and tts designed the experiments. cfl, tts, and ppz performed the experiments. cfl and tts analyzed the data. xp, ssc, and yy prepared the reagents. cfl, ml, and nko drafted the manuscript. all authors read and approved the final manuscript. conflict of interest the authors declare that they have no competing interests. ethical statement the animal experiment was approved by the committee on the ethics of animal of zhejiang university (zju20170667) and implemented in accordance with the animal care and ethics guidelines. this article does not contain any studies with human participants performed by any of the authors. engineering the largest rna virus genome as an infectious bacterial artificial chromosome construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis a recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype molecular characterization of feline infectious peritonitis virus strain df-2 and studies of the role of orf3abc in viral cell tropism completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus reverse genetics system for the avian coronavirus infectious bronchitis virus coronaviruses in poultry and other birds the long view: 40 years of infectious bronchitis research infectious bronchitis virus replication in the chicken embryo related cell line recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m41 strain remains attenuated but induces protective immunity production and characterization of monoclonal antibodies to poly100s1 protein of avian infectious bronchitis virus coronavirus spike protein and tropism changes identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus review of infectious bronchitis virus around the world recombinant infectious bronchitis coronavirus h120 with the spike protein s1 gene of the nephropathogenic ibyz strain remains attenuated but induces protective immunity the making of infectious viral rna-no size limit in sight reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus a reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted rna recombination binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication characterization of an avian infectious bronchitis virus isolated in china from chickens with nephritis establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h120 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-012064-egzl6zk9 authors: koopman, frank w.; de winde, johannes h.; ruijssenaars, harald j. title: c(1) compounds as auxiliary substrate for engineered pseudomonas putida s12 date: 2009-06-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-009-1922-y sha: doc_id: 12064 cord_uid: egzl6zk9 the solvent-tolerant bacterium pseudomonas putida s12 was engineered to efficiently utilize the c(1) compounds methanol and formaldehyde as auxiliary substrate. the hps and phi genes of bacillus brevis, encoding two key steps of the ribulose monophosphate (rump) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. this approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in c-limited chemostats fed with a mixture of glucose and formaldehyde. with increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (c-mol biomass/c-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. the rump-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. the presence of an endogenous methanol oxidizing enzyme activity in p. putida s12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (c-mol/c-mol) biomass yield. thus, by introducing two enzymes of the rump pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of p. putida s12 as a bioconversion platform host. the solvent-tolerant pseudomonas putida s12 is used as a platform for the bioconversion of sugars into substituted aromatic compounds (nijkamp et al. 2005; nijkamp et al. 2007; verhoef et al. 2007; wierckx et al. 2005) . these compounds are produced via central metabolite intermediates such as l-tyrosine and l-phenylalanine, the formation of which is directly linked to cellular growth. in such a process, a significant proportion of the available substrate is not used to form biomass and product, but utilized for the generation of free energy (atp and proton gradient) and reducing equivalents (nad(p)h). since the sugar substrate represents an important cost factor in bioproduction processes (schmid et al. 2001) , the addition of a cheap auxiliary catabolic substrate to generate free energy and/or reducing equivalents may significantly improve the economy of the production process. previous studies have shown that co-utilization of thiosulfate or c 1 compounds like formate and formaldehyde leads to an increased yield on the primary carbon source (baerends et al. 2008; bruinenberg et al. 1985; harris et al. 2007; masau et al. 2001 ). another c 1 compound that can be used as auxiliary substrate is methanol. being more reduced than formate or formaldehyde, methanol can yield more reducing equivalents per c-mole. since methanol can be derived from biomass via synthesis gas (chmielniak and sciazko 2003) it is a promising renewable auxiliary substrate for biotechnological processes. the first step in methanol metabolism is the oxidation to formaldehyde, via dehydrogenases or oxidases (anthony 1986; sahm 1977) . formaldehyde is extremely toxic due to non-specific reactivity with proteins and nucleic acids. therefore, rapid and efficient formaldehyde metabolization is a crucial step in the utilization of methanol. several pathways for the metabolism of formaldehyde have been described. methylotrophic yeasts may assimilate formaldehyde by the xylulose-5-phosphate cycle. in this pathway, formaldehyde is coupled to xylulose-5-phosphate and converted into dihydroxyacetone and glyceraldehyde-3-phosphate (ga3p) by a specialized transketolasedihydroxyacetone synthase (yurimoto et al. 2005a, b) . alternatively, formaldehyde is oxidatively dissimilated to form formate and eventually carbon dioxide and water (yurimoto et al. 2005a, b) . in bacteria, three formaldehyde metabolic pathways are known. like yeasts, also bacteria may oxidatively dissimilate formaldehyde to formate and co 2 . alternatively, formaldehyde may be assimilated via the serine cycle. in this pathway formaldehyde is coupled to l-glycine to form l-serine (l-ser). l-ser is subsequently metabolized via a cyclic pathway that ensures replenishment of l-glycine and permits a drain on glycerate-3-phosphate to generate biomass (chistoserdova et al. 2003) . the third bacterial formaldehyde metabolic route is the rump pathway ( fig. 1 ) . in this pathway, formaldehyde is coupled to ribulose-5-phosphate (ru5p) forming hexulose-6-phosphate (hu6p). hu6p is isomerized to fructose-6-phosphate (f6p), that can be further metabolized via the embden-meyerhof-parnas (emp) pathway, the entner-doudoroff (ed) pathway, or the pentose phosphate pathway (ppp). when f6p enters the oxidative part of the ppp (after isomerization to glucose-6-phosphate (g6p)), the rump pathway constitutes a cyclic oxidation pathway for formaldehyde: g6p yields ru5p and co 2 while generating two nadph. this cyclic oxidative dissimilation of formaldehyde has been described in, a.o., methylobacillus flagellatus kt (chistoserdova et al. 2000) . the rump pathway may also constitute a formaldehyde assimilation pathway (fig. 1) . in this case one-third of the f6p formed enters either the emp or ed pathway and is converted into ga3p and pyruvate. pyruvate is used for the production of cell constituents whereas ga3p and two-thirds of the produced f6p are used to regenerate ru5p by a combination of transketolase, transaldolase, and isomerization reactions (jakobsen et al. 2006; kato et al. 2006; large and bamforth 1988) . previously, formaldehyde has been successfully applied as auxiliary substrate in yeast; however, its high toxicity hindered efficient co-utilization (baerends et al. 2008) . expression of formaldehyde dehydrogenase (fld) and a fig. 1 assimilation and dissimilation pathways for formaldehyde in p. putida s12pjnnhp(t). 1 hexulose phosphate synthase, 2 hexulose phosphate isomerase, 3 hexose phosphate isomerase, 4 glucose-6-phosphate dehydrogenase, 5 6-phosphogluconate dehydratase, 6 2-dehydro-3-deoxy-phosphogluconate aldolase, 7 transketolase, 8 transaldolase, 9 pentose phosphate isomerase, 10 pentose phosphate epimerase; 11 6-phosphogluconate dehydrogenase, 12 formaldehyde dehydrogenase, 13 formate dehydrogenase. ru ribulose, hu hexulose, f fructose, g glucose, xu xylulose, r ribulose, e erythrose, su sedoheptulose, kdpg 2-dehydro-3-deoxy-6-phospho-d-gluconate, hcho formaldehyde. black arrows indicate the assimilatory rump pathway, dashed arrows indicate the dissimilatory rump pathway, gray arrows indicate the linear oxidation of formaldehyde to carbon dioxide formate dehydrogenase (fmd) from the methylotrophic yeast hansenula polymorpha in saccharomyces cerevisiae resulted in an increased biomass yield with formaldehyde as auxiliary substrate (baerends et al. 2008 ). an increased tolerance towards formaldehyde was obtained by introducing the fld and fmd genes. the aim of the present study was to develop and optimize a p. putida s12 strain capable of efficiently utilizing formaldehyde as auxiliary substrate. since p. putida s12 possesses genes encoding formaldehyde and formate dehydrogenase, it was expected that p. putida s12 has a basic endogenous capacity to oxidize formaldehyde to formate and co 2 . to further improve this capacity, additional formaldehyde metabolic pathways were constructed by expressing the two key enzymes of the rump pathway, 3-hexulose-6-phosphate synthase (hps) and 6phospho-3-hexulose isomerase (phi). biomass yields were determined in chemostat cultures for the engineered strain on different mixtures of glucose and formaldehyde. the rump pathway strain showed significantly improved performance over the control strain, achieving a biomass yield-on-glucose of 91% when using formaldehyde as auxiliary substrate. replacing formaldehyde with the renewable auxiliary substrate methanol resulted in a biomass yield-on-glucose of 84%. the bacterial strains and plasmids used in this study are shown in table 1 . luria broth (lb) (sambrook and russel 2001) or phosphate buffered mineral salts medium (mm; hartmans et al. 1989 ) were used as indicated. mm was supplemented with 20 mm glucose (mmg) unless otherwise stated. antibiotics were added as required at the following concentrations: ampicillin, 100µg/ml; gentamicin, 10µg/ml (mm), and 25µg/ml (lb). heterologous gene expression was induced by addition of 0.1 mm of salicylate unless otherwise stated. carbon-limited chemostat cultures were performed either in 1.0 l (working volume) fermentor using a biofloiic controller (new brunswick scientific) or in 0.7 l (working volume) fermentor using a bioflo110 controller. mm was used with 10 mm glucose, 10 mg/l gentamicin and 0.1 mm sodium salicylate. chemostats were inoculated with 50-ml overnight cultures on mmg. the dilution rate (d) was set at 0.05 h −1 . after 36 h, the d was increased to 0.2 h −1 (glucose/formaldehyde chemostats) or 0.1 h −1 (glucose/ methanol chemostats). the temperature was maintained at 30°c and the ph was maintained at 7.0 by automatic addition of 2 n naoh. the stirring speed was controlled by dissolved oxygen (do) concentration set at 15%. either air or a mixture of air and oxygen was supplied, using a m + w instruments d-5111 mass-flow controller (glucose/methanol chemostat), respectively, brooks mass-flow controllers (5850 e series and 5850 tr series) and a 0154 control unit (brooks). dissolved oxygen tension was continuously monitored with an inpro model 6810 probe. for preparation of formaldehyde solutions, demineralised water was heated to 100°c. during cooling paraformaldehyde (sigma-aldrich) was added to obtain a 6 m stock solution. a 5 m naoh solution was added to until the paraformaldehyde was completely dissolved. before use, the ph of the formaldehyde solution was set to ph 7 with a 5 m hcl stock solution. the final formaldehyde concentration was measured by hplc. the hps-phi genes were amplified from genomic dna of b. brevis s1 (atcc 12524) using forward (5′-gcctcga gggaatacacacatttgcttgtac-3′) and reverse (5′-cgtctagactatcgagattggcatgtc-3′) primers, designed on the published sequence of hps and phi (takashita and yasueda 2004) . the original bicistronic gene organization was maintained as well as the native ribosome binding sites. standard amplification methods were used with accuprime pfx polymerase (invitrogen). the resulting this study a ap r , gm r , ampicillin, and gentamicin resistance, respectively pcr fragment was a-tailed for 30 min at 68°c using supertaq (sphaeroq). the fragment was subcloned in pgem®-t easy (promega) yielding pgem-hp. after digestion with noti, the hps-phi fragment was isolated from agarose gel and ligated into noti digested and dephosphorylated pjnn(t), yielding pjnnhp(t) with the hps-phi bicistron under the control of the nagaa promoter. after verifying the correct orientation by restriction analysis, pjnnhp(t) was transformed to p. putida s12 using a gene pulser electroporation device, yielding p. putida s12pjnnhp(t). plasmid dna was isolated using qiaprep spin miniprep kit (qiagen). dna fragments were isolated from 0.8% agarose gels with qiaexii gel extraction kit (qiagen). nucleotide sequencing reactions were performed by mwg biotech ag. standard molecular cloning techniques were performed according to sambrook and russell (2001) . cell densities were measured at 600 nm (od 600 ) with a biowave cell density meter (wpa ltd). cell dry weight (cdw) was calculated from od 600 values where 1 od 600 unit corresponds to 0.465 g/l cdw. for calculation of cmol biomass, cdw was divided by 24 (roels 1983) . glucose concentrations were analyzed by hplc (waters) using an aminex hdp-87 n column with 0.01 m na 2 hpo 4 as the eluent and a refractive index detector, or using an ion chromatography (dionex ics3000 system). for hplc-ri, an aminex hpx-87 n (bio rad) column (300×7.8 mm) was applied using 0.01 m na 2 hpo 4 as the eluent. for ion chromatography a carbopac pa20 column was used with 10 mm naoh as the eluent. organic acids were analyzed by hplc-uv (agilent 1100 system) or ion chromatography (dionex ics3000). for hplc-uv, an aminex hdp-87h (bio rad) column (300×7.8 mm) was used with 0.008 n h 2 so 4 as the eluent at a flow rate of 0.6 ml/min. the diode-array detector was set at 210 nm. for ion chromatography an ionpac ice as6 column (9×250 mm) was used with 0.4 mm heptafluorobutyric acid as the eluent. methanol, formate and formaldehyde were analyzed by ion chromatography (dionex ics3000) using an ionpac ice as1 column (9×250 mm) with 100 mm methyl sulphonic acid as the eluent. in addition, formaldehyde was analyzed by hplc-uv (agilent 1100) after derivatization with 2,4-dinitrophenylhydrazine (dnph). equal volumes of dnph solution (0.5 mg/ml in 3% (w/v) phosphoric acid) and the sample were mixed and incubated for 10 min at 30°c prior to hplc analysis. an eclipse xdb-c8 column (4.6×15 mm) was used with a mixture of 45% acetonitrile and 55% water as the eluent at a flow rate of 1.2 ml/min. the diode-array detector set at 360 nm. protein expression was analyzed by sds-page. samples were obtained by centrifuging 1 ml of culture at an od 600 of 1.5 (mid-exponential growth phase) at 16.000×g. the cell pellet was resuspended in 300μl of loading buffer according to sambrook and russell (2001) and heated at 100°c for 10 min. after centrifugation at 16,000×g 20μl of supernatant was applied on the gel for analysis. routinely, 15% precast tris-hcl gels (bio rad) placed in a criterion™ electrophoresis cell (bio rad) were used. electrophoresis was performed in running buffer containing 0.025 m tris (ph 8.8), 0.192 m, glycine, and 3.5 mm sds in demineralised water as described by sambrook and russell (2001) at 130 v. a broad-range unstained protein standard (bio rad) was used as a marker. gels were stained using coomassie brilliant blue r-250 staining solution (bio rad). for preparation of cell extracts, 50-ml cultures were harvested at late logarithmic growth phase by centrifugation at 3,200×g for 15 min at 4°c. cell pellets were resuspended in 3 ml of 50 mm potassium phosphate buffer (ph 7.5). all samples were kept on ice during the preparation of extracts. cells were disrupted using a branson sonifier with a micro tip at a pulse mode, output set at 4 and effective output set to 60%. after three rounds of sonication (45 s of pulsing and 15 s pause), cell debris was removed by centrifugation at 3,200×g for 15 min at 4°c. supernatants were desalted using a pd10 column (ge healthcare) and used for enzyme assays. protein concentration was measured using bradford reagent (sigma-aldrich). hps-and phi enzyme activities were determined in a single combined assay as described previously (arfman et al. 1990; kato 1990; orita et al. 2006) . the final product formed by these two enzymes from formaldehyde and ru5p is f6p that is converted into g6p by glucose-6-phosphate isomerase. the formation of g6p was coupled to nadph formation via glucose-6-phosphate dehydrogenase that converts g6p into glucono-1,5-lactone-6-phosphate. the reaction mixture (0.95 ml) consisted of 50 mm potassium phosphate (ph 7.5), 5 mm mgcl 2 , 5 mm ribose-5-phosphate (ri5p; sigma-aldrich), 2.5 mm nadp (sigma-aldrich), 10 u phosphoribose isomerase (pri; sigma-aldrich), 10 u phosphoglucose isomerase (roche), 10 u glucose-6-phosphate dehydrogenase (sigma-aldrich). the mixture was preincubated for 5 min at 30°c to achieve equilibrium between ri5p and ru5p (catalyzed by pri). the reaction was started by adding formaldehyde to a concentration of 5 mm. for calculation of the combined hps-and phi activity, a molar extinction coefficient of 6.22 mm −1 cm −1 at 340 nm was used for nadph. one unit is defined as the overall hps and phi activity catalyzing the formation of 1μmol/min of nadph at 30°c. cloning and functional expression of hps and phi in p. putida s12 the thermotolerant methylotrophic bacterium b. brevis s1 was used as the source of the hps and phi genes encoding the first two enzymes of the rump pathway. previously, these enzymes have been successfully expressed in the nonmethylotrophic mesophile escherichia coli (yurimoto et al. 2002) . therefore, a similar approach was followed for p. putida s12. heterologous protein expression in the constructed rump pathway strain, p. putida s12pjnnhp (t), was confirmed by sds-page (not shown). as the individual activities are difficult to assess, the functionality of the expressed enzymes was confirmed by measuring their combined activity according to arfman et al. (1990) . the overall activity of the two conversions performed by these enzymes was 6,800 u/g protein in cell extract of midlog phase cells from p. putida s12pjnnhp(t). as expected, no activity was observed in the empty vector control strain (p. putida s12pjnnmcs(t)). formaldehyde as auxiliary substrate: c-limited chemostat cultures at a dilution rate of 0.2 h −1 p. putida s12pjnnhp(t) and an empty vector control strain were cultured in a c-limited chemostat with glucose as the primary carbon source and formaldehyde as auxiliary substrate. the chemostat feed was supplied at a dilution rate of 0.2 h −1 and contained a fixed concentration of carbon (60 mm) consisting of a mixture of glucose and formaldehyde the ratio of which was altered during the experiment. the experiment was started with 10 mm glucose (60 mm carbon) and no formaldehyde. after reaching steady state (i.e., after approximately 25 h) 6 mm of glucose-carbon (i.e., 1 mm of glucose) was replaced with 6 mm of formaldehydecarbon (i.e., 6 mm of formaldehyde). more glucose was replaced with formaldehyde in this step-wise fashion until wash-out of the chemostat cultures was observed. both the hps/phi-expressing p. putida s12pjnnhp(t) and the empty vector control strain reached steady state when up to 50% of the total carbon feed consisted of formaldehyde. no accumulation of glucose or its corresponding acid metabolites gluconate and 2-ketogluconate was observed, confirming that the cultures were c-limited. also, no formaldehyde or formate accumulated in the cultures of either strain, indicating the presence of endogenous formaldehyde and formate dehydrogenases in p. putida s12 by which these compounds were metabolized to completion. figure 2a shows the biomass yield-per-glucose (c-mol biomass/c-mol glucose) for both the empty vector control and the rump pathway strain. both strains showed a yield increase with increasing formaldehyde levels (fig. 2b) . however, the performance of the rump pathway strain was consistently superior to the performance of the control strain. the yield increase of the rump pathway strain relative to the yield increase of the control strain (fig. 2c) consistently shows that the introduction of the rump pathway genes approximately doubled the endogenous yield increase. this effect is independent from the relative formaldehyde concentration within the range tested. fig. 2 a biomass yield on glucose in chemostat cultures of strains s12pjnnhp(t) (white bars) and s12pjnn(t) (gray bars). y xs represents the biomass yield (c-mol biomass per c-mol glucose). b biomass yield increase (on glucose) as function of relative formaldehyde concentration in chemostat cultures of strains s12pjnnhp(t) (triangles) and s12pjnn (t) (squares). the biomass yield on glucose at 0% relative formaldehyde concentration was considered as the baseline yield. the baseline yield was assumed to be independent on relative formaldehyde concentration. c yield increase factor of strain s12pjnnhp(t) over strain s12pjnn(t) as function of relative formaldehyde concentration in chemostat cultures. data are averages of duplicate experiments; error bars denote the maximum deviation from the average of two independent experiments formaldehyde as auxiliary substrate: c-limited chemostat cultures at low dilution rate both the empty vector control strain and the rump pathway strain washed out in chemostats at d=0.2 h −1 when the fraction of formaldehyde was increased to 60% of total carbon. wash-out of chemostat cultures coincided with accumulation of formaldehyde and formic acid. this result suggests that both the endogenous and the engineered formaldehyde metabolic pathway activities were too low to cope with the formaldehyde feed applied. to investigate this hypothesis, chemostat cultures were performed at a lower dilution rate and, consequently, a lower formaldehyde feed rate that is more in agreement with previous studies on continuous culture experiments with methylotrophic bacteria on formaldehyde (arfman et al. 1992; de boer et al. 1990; mitsui et al. 2005) . the experiment was started at a dilution rate of 0.2 h −1 and a 7:3 (c-mol) glucose-to-formaldehyde ratio (60 mm total c). when steady state was reached, the dilution rate was set to 0.03 h −1 . the formaldehyde feed concentration was kept constant whereas the glucose feed concentration was lowered to 2 mm (12 mm c) to achieve a 4:6 glucoseto-formaldehyde ratio (c-mol; 30 mm c total). figure 3 shows that the empty vector control strain did not reach steady state under these conditions. formaldehyde and formate accumulated and wash-out was observed. in contrast, p. putida s12pjnnhp(t) reached steady state with a biomass yield on glucose of 91% (c-mol biomass/ c-mol glucose). since the rump pathway strain should theoretically be able to utilize formaldehyde as a sole csource (see fig. 1 ), the glucose feed was stopped. however, formaldehyde and formic acid accumulated and subsequent wash-out of biomass occurred, indicating that the assimilatory pathway activity for formaldehyde was insufficient to sustain growth at the conditions tested. methanol as auxiliary substrate: c-limited chemostat cultures at low dilution rate in the chemostat cultures performed in this study, formaldehyde (and formate) accumulated above a critical formaldehyde feed concentration, probably reflecting the upper limit of the formaldehyde metabolic capacity of the culture. accumulation of only trace amounts of formaldehyde was always accompanied by wash-out of the culture, underlining the acute and low-threshold nature of formaldehyde toxicity. thus, for the purpose of better process control, formaldehyde should be replaced with a less toxic auxiliary substrate such as methanol. utilization of methanol as (co-) substrate, however, requires the ability to oxidize methanol to formaldehyde. unexpectedly, formation of nadph was observed in hps/phi enzyme assays with p. putida s12 cell extracts when methanol was added as a substrate instead of formaldehyde. the overall activity amounted to 145 u/g of protein in cell extract. although the activity with methanol was lower by a factor 50 compared to formaldehyde, this result indicated that methanol is oxidized endogenously by p. putida s12. the c-limited chemostat cultures at d=0.03 h −1 were repeated, replacing formaldehyde with methanol. the chemostats were started at d=0.1 h −1 and a mixed feed of glucose and methanol was applied according to the following regime between the different steady states: glucose/methanol: 10 mm/15 mm; 5 mm/15 mm, and 5/30 mm. after the final steady state, the dilution rate was set at 0.03 h −1 and the total feed carbon concentration was decreased to 30 mm with 60% originating from methanol. p. putida s12pjnnhp(t) reached steady state under these conditions, in contrast to the empty vector control strain (fig. 4) . approximately 25% of the methanol feed was not metabolized, but no formaldehyde or formate accumulation was observed. the co-utilization of methanol resulted in a fig. 3 c-limited chemostat cultures of p. putida s12pjnnhp(t) (triangles) and p. putida s12pjnn(t) (squares) at d=0.03 h −1 , grown on mineral salts medium with 30 mm of total carbon (60% of all carbon originates from formaldehyde). the data presented are from a single representative experiment fig. 4 c-limited chemostat cultures of p. putida s12pjnnhp(t) (triangles) and p. putida s12pjnn(t) (squares) at d=0.03 h −1 , grown on mineral salts medium with 30 mm of total carbon (60% of all carbon originates from methanol). the data presented are from a single representative experiment significant improvement of the biomass yield to 84% biomass-per-glucose (c-mol). after stopping the glucose feed, the culture washed out as observed in the glucose/ formaldehyde experiments while culture methanol levels increased. discussion p. putida s12 was shown to have an innate capability to coutilize formaldehyde and glucose, in relative concentrations of up to 50% in c-limited chemostats. this ability is indicative of the presence of an endogenous formaldehyde metabolic pathway in p. putida s12. database searches revealed the presence of several genes coding for formaldehyde and formate dehydrogenases in the p. putida s12 genome (manuscript in preparation). therefore, this pathway probably consists of a linear route that oxidizes formaldehyde, via formate, to co 2 . the observed yield increase of the control strain may be attributed to the reducing equivalents generated during formaldehyde oxidation. although p. putida s12 disposes of an endogenous formaldehyde detoxification pathway, the introduction of an additional, heterologous formaldehyde metabolic pathway clearly had a positive effect in chemostat cultures grown on a mixture of glucose and formaldehyde. this was reflected by a consistently improved biomass yield on glucose and the ability to grow at elevated relative concentrations of formaldehyde. the presence of an additional formaldehyde metabolic route may allow for growth at higher relative formaldehyde concentrations, since accumulation of the highly toxic formaldehyde is prevented more efficiently. this unlikely explains, however, the higher biomass yield gain for the strain expressing the rump pathway enzymes. possibly, the improved yield gain relates to a better cofactor balance: when constituting a cyclic oxidation pathway, the rump cycle generates 2 mol of nadph per mol formaldehyde (fig. 1) , whereas the linear oxidation pathway yields 2 mol of nadh. it should be noted, however, that the introduction of the hps and phi genes will also constitute an assimilatory rump pathway (fig. 1) . thus, (co-)assimilation of formaldehyde may occur, in which case formaldehyde not only serves as a catabolic auxiliary substrate, but also as an assimilatory substrate. it was shown that the presence of the rump pathway results in a yield increase that is twice the yield increase attained by linear formaldehyde oxidation. considering that the same amount of formaldehyde was metabolized in both strains, it may be concluded that the rump pathway strain utilizes formaldehyde twice as efficient as the empty vector control strain. since the linear and the cyclic oxidation pathway yield an equal amount of reducing equivalents ( fig. 1 ; although of different nature), co-assimilation of formaldehyde is the most likely explanation for the observed additional yield increase found with the rump pathway strain. thus, the biomass yield expressed as yield-per-glucose likely is an overestimation of the actual biomass yield-per-assimilated carbon for the rumppathway strain grown on formaldehyde-glucose mixtures. the exact relative contributions of linear oxidation, cyclic oxidation, cofactor balance, and assimilation of formaldehyde to biomass yield cannot be determined as it is not possible to selectively shut down either of the pathways. attempts to knock-out formaldehyde dehydrogenase-encoding genes in order to abolish linear oxidation in p. putida s12 have consistently failed (unpublished) . no viable double crossover recombinants could be obtained, suggesting that the linear oxidation pathway apparently is crucial for the detoxification of formaldehyde. also in non-methylotrophs, formaldehyde is an endogenous metabolite that is produced by oxidative demethylation of dna (aas et al. 2003; falnes et al. 2002) and during methionine, histidine, and choline metabolism (arnstein 1954) . moreover, the contributions of the cyclic oxidation pathway and the assimilatory pathway cannot be separated since the expression of hps and phi constitutes both pathways at a time. despite the likely ability of p. putida s12pjnnhp to utilize formaldehyde as sole c-source, chemostat cultures washed out when deprived of glucose while maintaining the formaldehyde feed. the activity of the first two enzymes of the rump pathway as measured in mid-log phase batch cultures should suffice to assimilate the formaldehyde administered to the low-d (0.03 h −1 ) cultures. the net hps/phi activity amounted to approximately 6,800 u/g protein. assuming a protein content of 50%, this activity should allow 1 g of cells (dry weight) to cope with a formaldehyde feed of about 200 mmol h −1 . however, in the low-d cultures 270 mg of cdw was not able to fully metabolize formaldehyde that was administered at a rate of 0.54 mmol h −1 . this observation suggests that the metabolic flux through the endogenous part of the formaldehyde assimilatory cycle may be the bottleneck in preventing formaldehyde accumulation, and subsequent wash-out of cells, when deprived of other c-sources. alternatively, the activity of the rump-pathway enzymes in batch cultures may not be representative for chemostat cultures. replacement of formaldehyde with methanol in c-limited chemostat cultures at a low dilution rate also resulted in improved biomass yield. methanol was not completely utilized, however, and the biomass yield on glucose was slightly lower than for the corresponding formaldehyde experiments. the accumulation of methanol but not formaldehyde/formate suggests that the endogenous methanol dehydrogenase activity is the bottleneck for methanol utilization. this was also indicated by the 50-fold lower activity with methanol as the substrate in the hps/phi enzyme assay. from the p. putida s12 genome sequence (manuscript in preparation) no close homologues to established methanol dehydrogenases could be identified. therefore, it is likely that the low methanol dehydrogenase activity of p. putida s12 results from a side activity of a broadspecificity alcohol dehydrogenase. in conclusion, the enhanced ability of p. putida s12pjnnhp(t) to utilize c 1 compounds as auxiliary substrate can be used to substantially improve raw feedstock utilization efficiency. since both primary and auxiliary substrates can be obtained from biomass, process economy can be improved without compromising sustainability. although somewhat less efficient than formaldehyde, methanol is the preferred auxiliary substrate as it is less toxic, easier to handle, cheap and renewable. currently, possibilities are explored to improve the utilization of methanol by increasing the endogenous methanol dehydrogenase activity or by co-expressing a heterologous methanol dehydrogenase. human and bacterial oxidative demethylases repair alkylation damage in both rna and dna bacterial oxidation of methane and methanol 3-hexulose-6-phosphate synthase from thermotolerant methylotroph bacillus c1 environmental regulation of alcohol metabolism in thermotolerant methylotrophic bacillus strains the metabolism of glycine engineering and analysis of a saccharomyces cerevisiae strain that uses formaldehyde as an auxiliary substrate utilization of formate as an additional energy source by glucose-limited cchemostate cultures of candida utilis cbs 621 and saccheromyces cerevisiae cbs 8066 methylotrophy in methylobacterium extorquens am1 from a genomic point of view analysis of two formaldehyde oxidation pathways in methylobacillus flagellatus kt, a ribulose monophosphate cycle methylotroph co-gasification of biomass and coal for methanol synthesis regulation of methanol metabolism in the facultative methylothroph nocardia sp. 239 during growth on mixed substrates in batch-and continuous cultures alkb-mediated oxidative demethylation reverses dna damage in escherichia coli formate as an auxiliary substrate for glucose-limited cultivation of penicillium chrysogenum: impact on penicillin g production and biomass yield metabolism of styrene oxide and 2-phenylethanol in the styrene-degrading xanthobacter strain 124x bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase high-rate 3-methylcatechol production in pseudomonas putida strains by means of a novel expression system upregulated transcription of plasmid and chromosomal ribulose monophosphate pathway genes is critical for methanol assimilation rate and methanol tolerance in the methylotrophic bacterium bacillus methanolicus 3-hexulose-6-phosphate synthase from mycobacterium gastri mb19 the physiological role of the ribulose monophosphate pathway in bacteria and archaea methylotrophy and biotechnology mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown thiobacillus thiooxidans formaldehydelimited cultivation of a newly isolated methylotrophic bacterium, methylobacterium sp. mf1: enzymatic analysis related to c1 metabolism the solventtolerant pseudomonas putida s12 as host for the production of cinnamic acid from glucose optimization of the solvent-tolerant pseudomonas putida s12 as host for the production of p-coumarate from glucose the ribulose monophosphate pathway substitutes for the missing pentose phosphate pathway in the archaeon thermococcus kodakaraensis energetics and kinetics in biotechnology metabolism of methanol by yeast industrial biocatalysis today and tomorrow coryneform bacterium transformed to utilize methanol as carbon source bioproduction of p-hydroxybenzoate from renewable feedstock by solventtolerant pseudomonas putida s12 engineering of solvent-tolerant pseudomonas putida s12 for bioproduction of phenol from glucose hxlr, a member of the duf24 protein family, is a dna-binding protein that acts as a positive regulator of the formaldehyde-inducible hxlab operon in bacillus subtilis the ribulose monophosphate pathway operon encoding formaldehyde fixation in a thermotolerant methylotroph assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism acknowledgments the authors thank ton van maris for his expert help during the research of this paper, and nick wierckx for critical reading of this manuscript. this project is financially supported by the netherlands ministry of economic affairs and the b-basic partner organizations (www.b-basic.nl) through b-basic, a public-private nwo-acts program (acts = advanced chemical technologies for sustainability). this project was (co)financed by the kluyver center for genomics of industrial fermentation, which is part of the netherlands genomics initiative/netherlands organization for scientific research.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012058-ds7u3ke9 authors: verma, pradeep; dyckmans, jens; militz, holger; mai, carsten title: determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity date: 2008-08-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1525-z sha: doc_id: 12058 cord_uid: ds7u3ke9 beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (dmdheu) to increasing weight percent gains (wpg). the resistance of the treated specimens against trametes versicolor and coniophora puteana, determined as mass loss, increased with increasing wpg of dmdheu. metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (tea) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. both methods revealed that the fungal activity was related with the wpg and the mass loss caused by the fungi. still, fungal activity was detected even in wood blocks of the highest wpg and showed that the treatment was not toxic to the fungi. energy production showed a higher consistency with the mass loss after decay than tea; higher mass loss was more stringently reflected by higher heat production rate. heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide. chemical modification of solid wood is a novel technology to achieve protection of wood exposed to outdoor weathering conditions. the technology aims at enhancing the durability of wood in terms of resistance against wood decay fungi and insects. chemical modification can also improve various material properties of wood such as dimensional stability, compression strength, hardness, as well as weathering and uv stability (hill 2006) . the main processes which have been applied to chemically modify wood are acetylation (goldstein et al. 1961; militz 1991; rowell 2006; hill and jones 1999) , treatment with aldehydes or ketones (akitsu et al. 1993; yano and minato 1993; yasuda and minato 1994) and furfurylation (lande et al. 2004) . another very promising process is the treatment of wood with 1,3-dimethylol-4,5-dihydroxyethylene urea (dmdheu). in the preceding studies, wood modification with dmdheu was shown to increase resistance against decay fungi, to improve dimensional stability and to slightly reduce the moisture uptake of wood (militz 1993; yasuda and minato 1994; yusuf 1996; kabir et al. 1992) . the general principle of chemical wood modification is the reaction of a chemical agent with functional groups (mostly hydroxyl groups) of the cell wall polymers (cellulose, hemicellulose, lignin) and the formation of covalent bonds. this causes a change in the chemical and physical characteristics of wood (rowell 1983; norimoto 2001; hill 2006) . due to the introduction of bulkier groups within the cell wall, the dimensions of the cell wall increase and the pore size is reduced (hill and jones 1996; kwon et al. 2007) . as a result, the dimensional stability generally increases and the equilibrium moisture content decreases, since less space is available between the cell wall polymers to incorporate water molecules. the fibre saturation point of wood (approximately 25-35%) is an important factor for fungal colonisation of wood. fungi require wood moisture content above fibre saturation, because under these conditions free water is available in the lumens of the wood cells (eaton and hale 1993) . high resistance against fungal decay is assumed to be due to changes in the material properties of wood rather than a toxic effect on fungal physiology. this implies that modified wood can principally be colonised by decay fungi, since modified wood is not toxic to the fungi, but degradation of the modified cell wall is impeded (hill 2006) . studies on the resistance of modified wood to fungal degradation are almost exclusively based on standard test procedures which are limited to the determination of mass loss and/or loss of dynamic modulus of elasticity (moe). few studies have addressed the protection mechanisms of wood modification against decay fungi (ohkoshi et al. 1999; ritschkoff et al. 1999; papadopoulos and hill 2002; mohebby and militz 2002) . activity of decay fungi in wood is usually assessed by measuring the linear growth of the fungal hyphae or the mass loss of the infected wood. in addition, respiration, i.e. production of carbon dioxide (weigenand et al. 2008) , atp or ergosterol (bjurman 1992 (bjurman , 1994 has been used to evaluate microbial activity. in this study, determination of total esterase activity and micro-calorimetry were used to estimate the colonisation and biomass production on untreated and dmdheumodified wood. the determination of the total esterase activity of the fungi is based on hydrolysis of fluorescein diacetate (fda, 3,6-diacetoxyfluoran). fda is cleaved to fluorescein and acetic acid by different enzymes, such as proteases, lipases and esterases (guilbault and kramer 1964) . these enzymes are constituents of the primary metabolism of micro-organisms (swisher and carroll 1980) . released fluorescein can be quantified by fluorometry or spectrophotometry (schnürer and rosswall 1982; kerem et al. 1992; bjurman 1993) or localised by fluorescence microscopy (saxena and lysek 1993) . isothermal micro-calorimetry has been used as a nondestructive technique that requires only small sample volumes, is easy to handle and displays high reproducibility and sensitivity (criddle et al. 1991) . a micro-calorimeter determines the heat production rate (microwatt) in an adiabatic system, where heat is created by chemical reactions or the metabolic activity of living organisms. the total (net) heat output is equal to the metabolic enthalpy change and can be divided into a catabolic and an anabolic change (dermoun and belaich 1980) . catabolic metabolism, however, constitutes the main part of metabolism in terms of energy release. it was estimated that anabolic metabolism contributed 1.5% to the total heat production for oxic growth of yeast and bacteria and 8% for anoxic growth (belaich 1980; larsson et al. 1991) . the released heat is proportional to the consumption of nutrients as long as the mode of metabolism does not change (xie et al. 1997) . this technique has been used previously to determine the microbial activity in soils (vor et al. 2002; dyckmans et al. 2006 ) and of yeasts (ölz et al. 1993) . only a few studies describe the use of micro-calorimetry to determine the physiological activity of rotting fungi (ginterova and lazariva 1989; xie et al 1997; bjurman and wadso 2000) . heat production caused by fungi in an infected wood block can be directly related to the mass loss of the same block; for total esterase activity (tea) determination, however, the wood samples need to be crushed and degradation products leach out during the test in aqueous solution. therefore the mass loss after testing is difficult to determine. the brown rot fungus coniophora puteana (schum.: fr.) karst. strain bam ebw. 15 (dsm 3085) and the white rot fungus trametes versicolor (linneus) l. quélet strain ctb 863 a (dsm 3086) were obtained from the federal agency of material research (bam). the cultures were maintained on 4% malt extract agar medium at 22°c; sub-culturing was done twice a month to check virulence of test organisms on wood. mini-blocks (30 [longitudinal]× 10× 5 mm 3 ) of beech (fagus sylvatica l.) and pine sapwood (pinus sylvestris l.) were vacuum pressure impregnated at 100 mbar (1 h) and 12 bar (1 h) with aqueous solutions which contained 0.12 mol l −1 (7.3 mmol l −1 ), 0.24 mol l −1 (14.7 mmol l −1 ), 0.40 mol l −1 (24.9 mmol l −1 ), 0.83 mol l −1 (51.7 mmol l −1 ) and 1.77 mol l −1 (110.9 mmol l −1 ) of dmdheu (basf, ludwigshafen, germany) and of mgcl 2 ·6h 2 o (in brackets). after impregnation, the specimens were removed from the treatment solution, pre-dried at room temperature for 24 h and subsequently dried at 120°c for 24 h. the weight percent gain (wpg) was calculated from the dry masses before and after treatment as previously described (donath et al. 2004 ). twelve replicates were used per treatment (n=12). a mini-block test comparable with the european standard (1996) en 113 was carried out to assess fungal decay caused by basidiomycetes (bravery 1978) . dmdheu-treated and untreated specimens were stored in a climate chamber at 20°c and 65% relative humidity for 2 weeks in order to reach moisture equilibration. the blocks were sterilised by gamma radiation (25 kgy, isotron, netherlands). petri dishes (145 mm diameter) with malt extract agar (1.5% agar and 4% malt extract, 50 ml per plate) were inoculated with a mycelium agar disc (5 mm diameter) taken from the sub-margin of 1-month-old cultures of c. puteana or t. versicolor. when the fungal mycelium reached the border of the plate (approx. 14 days), three untreated and three modified mini-blocks were aseptically added on separate metal grids (to avoid moisture uptake by the wood block). the plates were incubated at 22±1°c at 65% relative humidity for 12 weeks. twelve wood samples for each treatment from four different plates were used to determine the mass loss of the wood blocks. wood mini-blocks which were incubated with wood decay fungi over different time periods (3, 6, 9 and 12 weeks) were taken from the plates, and the surface mycelium was removed. the blocks were crushed with a mortar and a pestle to small pieces (under ice cooling) and dispersed in 100 ml of sterile 60 mmol l −1 sodium phosphate buffer solution (ph 7.6) in an erlenmeyer flask (500 ml). a stock solution of fluorescein diacetate (fda, 3,6-diacetoxyfluran, c 24 h 16 o 7 ; sigma chemical co., st. louis, usa) in acetone (2 mg ml −1 ) was added to reach a final fda concentration of 10 μg ml −1 . the flasks were incubated at room temperature on a rotary shaker (120 rpm) for 1 h in total. after intervals of 15 min, 1 ml aliquots were taken from the flasks and placed in eppendorf tubes. the hydrolysis of fda was terminated by addition of acetone to reach a final acetone concentration of 50% (v/v; schnürer and rosswall 1982) . the mixture was centrifuged for 5 min in a micro-centrifuge (iec micromax, netherlands) at 10,000 rpm to remove suspended particles. the amount of fluorescein released was measured (specord 205, analytic jena, germany) at the absorbance maximum of 494 nm; esterase activity was expressed as milligram fluorescein which was released from fda within 60 min (kerem et al. 1992; swisher and carroll 1980; schnürer and rosswall 1982) . three individual wood blocks were used for each treatment to determine the activity. heat production was measured with a four channel microcalorimeter (thermal activity monitor 2277, thermometric, jarvalla, sweden) of heat conduction type and recorded using the program digitam 2.0. wood mini-blocks were incubated with wood decay fungi according to bravery (1978) as described above. after different incubation times (3, 6, 9 and 12 weeks), the wood samples were removed from the petri dish and placed into a measuring ampoule (25 ml) in order to determine the heat production. three types of handling prior to the measurement were tested to assess if the method of sample removal from the agar plate has an effect on heat production. for the first method, the surface mycelium of each wood block was aseptically removed with a scalpel. for the second method, the same specimens were kept in sterile petri dishes for 7 days at room temperature to allow the fungal mycelium to recover from primary injury. for the third method, the incubated samples were taken from the petri dishes by cutting an agar block around the sample to minimise damage to the fungal hyphae. the sample was inserted into the measuring ampoule together with the agar block. for the studies presented in figs. 5 and 6, the first method was applied. during one measurement, heat production of three samples was simultaneously recorded; an empty ampoule served as a reference ampoule; un-inoculated wood did not produce detectable heat in the test. the three samples comprised one replicate for a specific treatment. three replicates per treatment were tested in total. all measurements were performed at 25°c; during the measurement, the micro-calorimeter was located in a climatic chamber at constant temperature. internal calibration was carried out at 3,000 μw in the static mode. the detection limit was ±9 μw and baseline stability over 24 h was ±15 μw. the ampoules were allowed to equilibrate for 1 h in the stand-by position before heat production data were recorded in intervals of 300 s over 20 h. after the measurement, the samples were dried at 103°c and weighed. the amount of oxygen in the ampoules at the beginning of the measurement was calculated to be approx. 0.21 mmol. oxygen consumption during the measurements was calculated based on the energy production according to wieser (1986) . cumulative energy production (e total ) [j] was calculated from the following equation: where q i = heat [μw] produced per time interval; t = time interval (300 s). beech and scots pine wood blocks linearly increased in weight percentage gain after treatment with increasing concentrations of dmdheu. pine specimens displayed higher weight gains at a given concentration compared to beech due to their lower density (table 1) . during the decay test, all wood blocks were overgrown by surface mycelium independent of the wpg of the wood. increasing wpg from dmdheu treatment significantly decreased the mass loss in both beech and pine caused by the white rot fungus t. versicolor and the brown rot fungus c. puteana after fungal incubation (fig. 1) . in beech, mass losses of the controls were greater than 60% after 12 weeks of incubation reflecting high activity of the tested fungi. at moderate and high wpg (approx. 13% and 28%), the mass loss in beech specimens was less than 6% with t. versicolor and less than 10% with c. puteana, while at low wpg (approx. 0.8% to 6.8%), higher mass loss was observed for t. versicolor (approx. 45%) and c. puteana (18-26%). mass losses of pine sapwood controls were lower than those of beech wood controls, particularly with t. versicolor. as observed for modified beech wood, pine specimens with moderate and high wpg were minimally degraded by the fungi. in contrast to the degradation of beech wood, pine with low wpg (1.3%) provided significant resistance against t. versicolor (6% mass loss), while c. puteana caused mass loss of 26%. total esterase activity tea of t. versicolor in untreated beech wood was higher than that of c. puteana in untreated beech (fig. 2a,b) . t. versicolor promoted the highest fluorescein release of 60 μg ml −1 in untreated beech (after 6 weeks), while the maximum fluorescein release produced by c. puteana was only half of that of t. versicolor. specimens with wpg between 3% and 10% incubated with t. versicolor displayed lower tea than the controls. at high wpg, the activity of t. versicolor was barely above the detection limit. tea of c. puteana in treated beech was less affected by wpg with mass loss due to decay being less than that for t. versicolor. similar results were obtained for the pine specimens (fig. 2c,d) . c. puteana showed the tendency that higher wpg was concomitant with lower tea; however, the differences between the treated specimens and the control were small, as with c. puteana in beech. t. versicolor in pine, however, showed considerable differences between the treated specimens and the control. the activity of all treated samples was close to the detection limit. thus, tea activity differences between them were not detectable. in the pine controls incubated with t. versicolor, tea (fig. 2c) continuously increased for 12 weeks. with all other fungi and wood species, maximum values of tea were reached between 6 and 9 weeks. the thermograms of t. versicolor in untreated and modified beech wood (fig. 3a) displayed typical courses of heat production after 3 weeks of incubation on agar plates. heat production started immediately showing a maximum after approx. 2 h. in the untreated controls, the rate of heat production decreases quickly after that peak, while it remained almost constant in the treated samples. as a consequence, total energy production, which was calculated from the cumulative heat production, did not proceed linearly over longer measurement periods when heat production was high (fig. 3b) . on average, only 40% of the initial oxygen in the ampoules with untreated beech wood remained after 17 h (fig. 3c) . the removal of mycelium from the wood surface injured the hyphae potentially inducing primary metabolic activity of the fungi. in most cases, energy production was higher when the wood blocks were removed together with the agar which potentially damaged less of the mycelia (fig. 4) . however, the agar would also have included more of the fungal biomass and this could also explain the larger energy values. in pine, the energy production of specimens incubated for 7 days after removal of surface mycelium was reduced by up to 46% compared to measurements taken directly after removal of mycelium (after 12 weeks of fungal incubation); these differences, however, were less for the samples measured after 6 weeks of incubation. the greatest differences were observed with c. puteana in beech. in this case, however, energy production after removal of mycelium displayed high standard deviation. as with total esterase activity, t. versicolor produced more heat energy in beech (fig. 5a ) than in pine wood with mycelium (directly measured) fig. 4 total energy production of decay fungi in wood mini-blocks during 3 h measuring time. heat production was recorded directly after removal of surface mycelium from the wood block, 7 days after the removal of surface mycelium, without removal of surface mycelium (wood block still placed on an agar block that after incubation was cut from the medium around the sample to minimise damage to the fungal hyphae). a 6 weeks of incubation, b 12 weeks of incubation (error bars show standard deviation; three replicates were used per treatment) (fig. 6a) . energy production, however, did not always follow the same course as tea. t. versicolor in untreated beech and in beech treated with low dmdheu concentration (fig. 5a) produced similar amounts of energy as c. puteana in the corresponding specimens (fig. 5c) . in contrast, tea of t. versicolor in untreated beech (fig. 2a) was much higher than that of c. puteana in corresponding specimens (fig. 2b) . a comparison of both fungal species in the untreated beech samples revealed that t. versicolor and c. puteana (fig. 5b,d) degraded the wood at comparable rates. in the pine specimens, c. puteana reached a maximum mass loss total energy production of decay fungi in untreated (control) and dmdheu treated (0.12-1.77 mmol l −1 dmdheu solution) pine mini-blocks during 3 h measuring time. after the indicated incubation times on malt agar plates, the surface mycelium of each wood block was aseptically removed and the activity in the blocks directly determined. a energy production and b mass loss caused by t. versicolor; c energy production and d mass loss caused by c. puteana (error bars show standard deviation; three replicates were used per treatment) (50%) after 6 weeks (however, the standard deviation was high), yet energy production continually increased from 6 to 12 weeks of incubation (fig. 6c,d) . overall, the energy production of both fungi in beech and pine tended to decrease with increasing weight gain of dmdheu and, thus, reflected the differences in mass loss during fungal incubation (figs. 5 and 6 ). specimens treated with 0.12, 0.24 and 0.40 mol l −1 of dmdheu displayed considerable energy production with both fungi and wood species, while higher treatment concentrations (0.83 and 1.77 mol l −1 ) reduced energy production significantly. c. puteana showed higher energy production in high wpg (0.83 and 1.77 mol l −1 ) beech and pine samples compared to t. versicolor. this reflected the higher mass loss caused by c. puteana (compare fig. 5b and d, as well as fig. 1a and b) in high wpg samples and this is also in agreement with the tea data. decay of beech and pine sapwood by t. versicolor and c. puteana was strongly affected by dmdheu treatment. except for t. versicolor in beech, even treatment with the lowest concentration of dmdheu (0.12 mol l −1 ) reduced the mass loss of all specimens by more than 50% compared to the controls. in beech, treatment with low dmdheu concentrations provided increased protection against c. puteana (lower mass loss) than against t. versicolor, while in pine, dmdheu was more effective against t. versicolor. this effect might be explained by the preference of brown rot fungi for softwoods and of white rot fungi for hardwoods (eaton and hale 1993) . beech wood is a more natural habitat for t. versicolor than pine. therefore, the fungus can obviously cope with higher levels of dmdheu treatment in beech than in pine wood. c. puteana, on the other hand, did not show significant differences in mass loss in comparing beech and pine. dmdheu monomers were recently shown not to have a negative effect on the growth of the two fungi tested, when the chemical was added to agar growth medium (verma et al. 2005) . the lack of toxicity of dmdheu has also been shown by its rather high ld50 (>10,000 mg kg −1 ) for rats (oecd sids 2000) . thus, a biocidal effect of unreacted monomers cannot explain the reduction in mass loss. however, the modification of beech and pine wood with dmdheu to a wpg greater than 10% provided a level of protection against t. versicolor and c. puteana, comparable to that of conventional wood preservatives. increasing the wpg of dmdheu also resulted in lower metabolic activity of the infecting fungi. energy production and tea were observed at the highest wpg, and this metabolic activity emanated from fungal hyphae within the wood blocks as the surface mycelium was removed prior to measurement. these results show that the fungi were not only able to overgrow, but also to colonise the wood blocks. the ability to colonise the wood, was also confirmed through microscopy which showed the presence of hyphae in the cells of modified wood (not shown). at high wpg, the fungi are able to grow into the wood and use readily available nutrients such as simple sugars, minerals, proteins or vitamins in the parenchyma cells. the degree of colonisation, however, remains low, since the fungi are not able to make nutrients accessible by degradation of cell wall polymers. in addition, the fungi do not seem to be able to transport sufficient amounts of nutrients from the outer agar medium into the wood. three main mechanisms have previously been cited to explain high resistance of chemically modified wood against fungal decay (hill 2006) : reduction of the moisture content (fibre saturation point), changes of the cell wall polymers to make these polymers unrecognisable for enzymes and/or a lower micro-pore size in the wood cell wall. it is assumed that cell wall bulking and micro-pore blocking can effectively inhibit the penetration of low molecular weight diffusible agents, which is required for fungal degradation (papadopoulos and hill 2002; hill et al. 2005) . the esterase activity and heat production did not always follow the same trends. in both fungi, the energy production appeared to be rather constant over the total incubation time (12 weeks), while fda hydrolysis remained variable. this could be the result of a higher systematic error, e.g. related to enzyme extraction during tea determination. the colonisation of the wood specimens was not confirmed by mass loss determination because the procedure required crushing and destruction of the samples. injury during the removal of mycelium from the wood surface did not seem to induce the metabolic activity (e.g. heat production) in the fungi. heat production was higher, when the injury was minimised by removing agar together with the wood block (still, considerable injury of hyphae occurred in the agar block). this might be explained by greater fungal biomass in the agar. specimens kept under sterile conditions to allow the mycelium to recover from injury showed a reduced activity. this could also be explained as the effect of non-optimal storage conditions (drying and surface death of mycelial cells), rather than a reduction in activity after remediation of the injury. during calorimetry, fungal activity was strongly affected by respiration, particularly at higher energy production levels. it was previously reported that high carbon dioxide concentrations affect fungal activity stronger than a lack of oxygen (scheffer 1986; ljungholm et al. 1979; sparling 1983) . determination of total esterase activity and metabolic heat (energy) in a micro-calorimeter are suitable methods for determining the fungal activity in decaying wood. both methods provide measures of fungal primary metabolism; however, secondary metabolism also contributes to heat production. micro-calorimetry, in particular, is a nondestructive method for rapid screening of wood preservatives 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cross-linking formation and its application to the reconstituted wood products acknowledgements the authors are grateful to the german science foundation (dfg) for supporting this project. they would also like to thank prof. jody jellison and prof. barry goodell for fruitful discussions and for their sound advice.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012435-tt44dkqd authors: temudo, margarida f.; muyzer, gerard; kleerebezem, robbert; van loosdrecht, mark c. m. title: diversity of microbial communities in open mixed culture fermentations: impact of the ph and carbon source date: 2008-10-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1669-x sha: doc_id: 12435 cord_uid: tt44dkqd anaerobic fermentation by an open mixed culture was investigated at different ph values (4–8.5) and with three substrates (glucose, glycerol and xylose). the populations established in each condition were assessed by denaturing gradient gel electrophoresis analysis of the 16s ribosomal rna gene fragments. the fermentation pattern and the composition of the microbial population were also evaluated when operational variations were imposed (increase of substrate concentration or introduction of a second substrate). the experimental results demonstrated that at low and high ph values, a clearly different fermentation pattern was associated with the dominance of a specialised group of clostridiae. at intermediate ph values, the product spectrum was rather variable and seemed to be sensitive to variations in the microbial community. different substrates resulted in the establishment of different microbial communities. when fed with a mixture of two substrates, mixotrophic microorganisms (capable of degrading both substrates) were found to overgrow the originally dominant specialists. overall, the experiments have shown that some operational variables have a clear impact on the fermentation pattern and on the population established. however, a uniform relationship between the process characteristics (associated to a metabolic response) and the microbial population present is not always possible. electronic supplementary material: the online version of this article (doi:10.1007/s00253-008-1669-x) contains supplementary material, which is available to authorized users. mixed culture biotechnology may become an attractive addition to traditional pure-culture-based biotechnology for the production of bulk chemicals from waste streams (dai et al. 2007 ; kleerebezem and van loosdrecht 2007; reis et al. 2003; rodriguez et al. 2006) . open mixed cultures, based on natural inocula with a high microbial diversity, allow continuous operation of bioprocesses under non-sterile conditions with no risk of strain degeneration. additionally, due to the high microbial diversity, these cultures can deal with mixtures of substrates and of variable composition. the conditions in these processes are chosen such that the metabolic conversion of interest provides an ecological advantage to the microorganisms. it has been proposed that in mixed culture fermentation (mcf) systems, the operational conditions will determine which catabolic product allows the more efficient growth and will therefore dominate (claassen et al. 1999; hawkes et al. 2007; rodriguez et al. 2006) . ecosystem functioning often depends on the microbial composition. recent studies on bacterial communities showed that changes in ecosystem functioning are associated with changes in the genetic structure of bacterial communities, suggesting that the performance of ecosystem-based processes depends on the bacterial community composition (dejonghe et al. 2001; wittebolle et al. 2005) . however, it has also been shown that bacterial communities are functionally redundant, i.e. communities with different compositions can perform similar functions such as catalysing metabolic reactions and primary production (fernandez et al. 2000; findlay and sinsabaugh 2003; langenheder et al. 2005) . the ph is a powerful parameter that can be used to control both the metabolism and to select the microorganisms that are best able to survive. the ph affects several microbial parameters such as the growth rate, the utilisation of the carbon source, efficiency of the substrate conversion, etc. (russell 1992) . furthermore, the ph determines the fraction of undissociated acids in the broth, which are known to be able to permeate cell membranes. indeed, it has been shown that the environmental ph has a strong impact on the product spectrum of mixed cultures fermentation (temudo et al. 2007; zoetemeyer et al. 1982) . another important factor that shapes the community structure and product formation is the substrate. the degree of reduction of the substrate and the metabolic pathway used for its degradation determine the product spectrum (temudo et al. 2008) . the most abundant sugar in nature, glucose, is always accompanied by other compounds, e.g. xylose. the growth on such mixtures may not be controlled by only a single nutrient, and the kinetic properties of a cell may change due to adaptation (kovarova-kovar and egli 1998) . to which extent these factors have an impact on the microbial community composition or on its functioning is an important question in microbial ecology. insight in these interactions between the environment and the microbial population may allow the prediction of the product spectrum formed in mcf systems. this is a prerequisite for successful implementation of mixed culture biotechnology-based processes. the fundamental question to be answered is whether the process conditions are determining the product spectrum or is the product spectrum resulting from the established microbial community. the aim of this research was to study the link between the functional performance and the microbial community found under each condition. furthermore, it was of interest to evaluate the response and stability of a certain population towards the introduction of other variables (product concentration or other carbon source). to this end, the microbial population established was assessed in several continuous culture fermentation systems operated under different operational conditions. the variables investigated were the ph and diverse carbon sources (glucose, glycerol and xylose). after achieving a stable performance, the impact of higher inflow substrate concentrations and introduction of a second substrate on the microbial community present was assessed. a continuously stirred tank reactor was operated at 30°c at an influent substrate concentration of 4 g/l. the bioreactor had a working volume of 2 l and was sparged with nitrogen gas to maintain anaerobic conditions. the reactor was freshly inoculated before each experiment with 40 ml of two different inocula (a sludge from a distillery wastewater treatment plant and another from a potato starch processing acidification tank, temudo et al. 2007) and was left in batch mode until the substrate was depleted. it took 3-5 weeks to establish stable operation. a stable operation was assumed when the measured product and biomass concentrations varied less than 20% within a time frame of at least a week. for the study of the impact of the ph on fermentation by mixed cultures, glucose was used as substrate. the ph values were investigated in the following order: 5.0, 4. 0, 5.5, 8.5, 7.75, 6.25, 4.75, 7 .0 and 8.0. the dilution rate was 0.05 h −1 for ph values equal or lower than 5.5 and 0.12 h −1 for ph values equal or higher than 5.5. this corresponded to approximately 30 volume changes at a dilution rate of 0.05 h −1 (ph≤5.5) and about 70 volume changes when the dilution rate was 0.12 h −1 (ph≥5.5). the influence of different substrates (glucose, glycerol and xylose) on the product spectrum and the composition of the microbial community was studied at ph 8.0, 30°c, and d 0.12 h −1 . the influent substrate concentration initially was 4 g/l and later increased to 10 g/l. in the case of the glucose and glycerol cultivated cultures, also 25 g/l was tested (temudo et al. 2008) . the mineral medium composition was described previously (temudo et al. 2007 ) and, when needed, was adjusted to keep the c/n/p ratio constant. fermentation of a mixture of two substrates was tested on the biomass cultivated on a single substrate. a mixture of glucose and glycerol was added to the glucose grown culture. the same substrates mixture was fed to the glycerol grown culture. a mixture of glucose and xylose was supplied to the reactor cultivated on xylose. in these experiments, the total amount of carbon supplied was kept constant, and the two substrates were fed in equal amount of carbon moles. the reactors were opened and cleaned at least once per week to remove wall growth and only allow suspended biomass to remain. the carbon source solutions were sterilised at 110°c for 20 min to avoid microbial growth in the vessel. reactor broth samples were immediately filtered (millipore membrane of 0.45 μm). the substrate and end products were determined and quantified. xylose, glucose, glycerol, volatile fatty acids (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and caproate), lactate, succinic acid, formic acid and 1,3-propanediol were determined. measurements of h 2 and co 2 in the off gas were performed online, and the base added to maintain the ph constant was monitored. a detailed description of the analytical methods used can be found elsewhere (temudo et al. 2007 ). the biomass dry weight was determined after filtration according to standard methods (greenberg et al. 1992) . nucleic acid extraction bioreactor community samples were concentrated by centrifugation. genomic dna was extracted directly from the concentrated biomass using the ultra clean soil dna extraction kit (mobio laboratories, california, usa) according to the manufacturer's protocol. extracted dna was stored at −20°c until further use. pcr amplification amplification of 16s ribosomal rna (rrna) gene fragments was performed using the primer pairs, 341f-gc (5′ cct acg gga ggc agc ag 3′) and 907r(5′ ccg tca att cmt ttg agt tt 3′; muyzer et al. 1995) . for the amplification reactions, 1 μl of genomic dna was used. the protocol used for the amplification of 16s rrna gene fragments was as described previously (muyzer et al. 1995) . polymerase chain reaction (pcr) amplification was performed in an automated thermal cycler. initial denaturation was at 94°c for 5 min; followed by 30 cycles of denaturation at 94°c for 1 min, annealing at 65°c for 1 min and extension at 72°c for 20 s, except for the last cycle which was 15 min. a touchdown protocol was used to increase specificity of the amplification reaction. this consisted of a decrease of 0.5°c in each cycle of the annealing temperature, 65°c to 55°c in 20 cycles. the quality of the pcr products was examined on 1% (wt./vol.) agarose gel, and the yield was quantified by absorption spectrophotometry using the nanodrop nd-1000 tm (nanodrop technologies, delaware, usa). dgge of 16s rrna gene fragments denaturing gradient gel electrophoresis (dgge) was performed as described by schäfer and muyzer (2001) using the d-code system (bio-rad laboratories, california, usa). electrophoresis was performed with 1-mm-thick 6% polyacrylamide gels (ratio of acrylamide to bisacrylamide, 40:1) submerged in 1× tae buffer (40 mm tris, 40 mm acetic acid, 1 mm edta, ph 7.5) at constant temperature of 60°c. pcr product in an amount ranging from 300 to 500 ng was applied to the individual lanes on the gel. the electrophoresis conditions for 16s rdna fragment were the same as described previously (schäfer and muyzer 2001) : 16 h at 100 v in a linear 30% to 60% denaturant gradient [100% denaturants is a mixture of 7 m urea and 40% (vol./vol.) formamide]. after electrophoresis, the gels were incubated for 30 min in milli-q water containing ethidium bromide (0.5 μg/ml), rinsed for 20 min in milli-q water and photographed using a bio-rad geldoc station (bio-rad). individual bands were excised, resuspended in 15 μl of milli-q water and stored overnight at 4°c. a volume of 3 to 5 μl of the supernatant was used for re-amplification with the original primer sets. the re-amplified pcr products were run again on a denaturing gradient gel to check their purity. prior to sequencing, the pcr products were purified using the qiaquick pcr purification kit (qiagen gmbh, hilden, germany). phylogenetic analysis the obtained 16s rrna gene sequences were first compared to sequences stored in genbank using the blast algorithm. subsequently, the sequences were imported into the arb software program (ludwig et al. 2004; schäfer and muyzer 2001) and aligned using the automatic aligner function. the alignment was further corrected manually, and an optimised tree was calculated using the neighbour-joining algorithm with felsenstein correction. sequence accession numbers the 16s rrna sequences determined in this study were deposited in genbank under accession numbers eu435415-eu435434. the ph not only had a significant effect on the product spectrum but also on the dominant type of microorganism as can be seen in fig. 1a ,b. the full product distribution as a function of the ph can be found in table s1 of the electronic supplementary material. at low ph values, the product spectrum mainly consisted of butyrate and acetate, while at high ph, it shifted to acetate and ethanol. the microbial population that established at each ph value also differed as can be seen from the dgge gel in fig. 1 . from the gel, the dominant bands were excised and re-amplified. the successfully sequenced dna fragments are numbered in fig. 1 and the respective phylogenetic affiliation is illustrated in the phylogenetic evolutionary tree (fig. 2) . four microbial communities could be distinguished: at low (4-5.5), medium (6-7) and high ph values, 7.5-8 and 8.5. the dominant microorganisms at low and at high ph were located at two different subclusters of cluster i of the genus clostridium. at middle ph values, the principal microorganism belonged to genus klebsiella; facultative anaerobic bacteria that are known for lacking the enzymes involved in butyrate production. this relates to the observation that at ph 6.25 and 7.0, the butyrate yield was lower (fig. 1) . to study and compare the effect of different substrates (glucose, xylose and glycerol), reactors were operated at ph 8, dilution rate 0.12 h −1 and substrate concentration 4 g/l (0.14-0.15 cmol/l). the system was characterised after 80 volume changes in the case of glucose and glycerol and after 240 volume changes for xylose. the main catabolic products of glucose and xylose fermentation were acetate, butyrate and ethanol (fig. 3) . the complete product spectrum can be found in table s2 of the electronic supplementary material. glycerol has a higher degree of reduction compared to glucose and xylose, and therefore, a higher fraction is converted to more reduced compounds, e.g. ethanol and 1,3-propanediol. in a chemostat at low substrate concentrations, the main product was ethanol, while minor amounts of 1,3-propanediol and acetate were produced (fig. 3) . the microbial populations that established on the three substrates were different ( fig. 2 and 4) . the glycerol and xylose cultures had a bigger diversity compared to the glucose grown culture. the glycerol community was dominated by an enterobacteria closely related to klebsiella oxytoca (band number 58), a facultative microorganism which has been reported to be able to ferment glycerol (homann et al. 1990 ). other microorganisms in the glycerol-grown culture were related to pectinatus frisingensis (band 61), to clostridium intestinale, clostridium cluster i (band 57), and the sequence from band 53 was related to uncultured bacteria of the cluster xiva of the clostridia family. species of the genus pectinatus have been reported in spoiled beer and are ethanol-tolerant up to 4.8%. these species belong to the clostridia family, cluster ix, and are strictly anaerobic (haikara and helander 2006) . in the bioreactor fed with xylose, there were three dominant bands present, represented by numbers 9, 18 and 23. the two bands indicated with the number 18 seem to be an artefact of this dna sequence. several attempts were made to purify; however, the result was always the same (two bands in the dgge gel) and the purified dna could be fig. 1 impact of the operational ph on glucose fermentation: a the product spectrum and b dgge analysis of the 16s rrna gene fragment of dna samples from the biomass present in the reactor at each condition. the dilution rate was 0.05 h −1 for the first ph values (4-5.5) and 0.12 h −1 for the last operational ph (5.5-8.5). symbols correspond to the yields of the main products (cmol/cmol): butyrate (square), acetate (circle) and ethanol (triangle). apart from biomass, the main other products were co 2 and h 2 at low ph values and formate at high ph values fig. 2 phylogenetic tree based on 16s rrna gene sequences obtained from the dgge bands. the sequences were mainly affiliated to a two distinct classes: gammaproteobacteria and bacteroidetes and to b three different genera of clostridia familiae (collins et al. 1994) successfully sequenced. the sequence 18 was affiliated to cluster i of the clostridium genus. the sequences 9 and 23 were affiliated to cluster xiva of the clostridia family. many of the described species of this cluster live in ruminant environments, which may explain a higher affinity for xylose. impact of the increase of the substrate concentration after characterising the system at low substrate concentrations in the influent, the substrate concentration of the feeding solution was increased. the system was characterised after reaching stable operation as obtained after approximately 40 volume changes. the main product yields are shown in fig. 3 ; full product distribution can be found in table s3 of the electronic supplementary material. the substrate concentration inside the reactor was below the detection limit. however, under the new conditions, higher substrate conversion rate and higher product concentration, the product spectrum shifted in the three reactors. in the reactor fed with glucose and xylose, butyrate decreased to very low values and ethanol and acetate became the major products (1:1). in the glycerol-fed reactor, the ethanol yield decreased, while 1,3-propanediol and acetate yields increased (fig. 3) . figure 4 shows the dgge pattern of the microbial community at elevated substrate concentrations. even though a similar shift in the catabolic product spectrum was observed in the glucose-and xylose-fed reactors, it only resulted in a clear change in the microbial population in the xylose-fed reactor. in the glucose system, the main microorganism remained. in the chemostat fed with glycerol, only small changes in the microbial community occurred, band 57 disappeared and 48 emerged. the cultures cultivated on different substrates (glucose, glycerol and xylose) were submitted to a mixture of two substrates: the glucose and glycerol grown cultures were fed with a glucose-glycerol mixture, and the xylose grown culture was fed with a xylose-glucose mixture. after 1 week feeding with the mixture of substrates, the product spectrum in all reactors was very similar to the average of the product spectra observed when the two substrates were fed separately (fig. 5) . the full product distribution can be found in table s4 of the electronic supplementary material. immediately after switching from a single substrate to a mixture of substrates, the populations present in the reactor were capable of degrading the additional substrate. the dgge pattern of the microbial communities at different stages is shown in fig. 6 . samples were taken before feeding with a mixture of substrates (lane i), 1 day from the bioreactors cultivated on different substrates: glucose, glycerol and xylose. impact of the substrate concentration on the microbial community: i glucose 4 g/l, ii glucose 26 g/l, iii glycerol 4 g/l, iv glycerol 23 g/l, v xylose 4 g/l and vi xylose 11 g/l after (lane ii) and 1 week after (lane iii). the glucosegrown culture could instantaneously convert glycerol. the composition of the microbial population was not affected after 1 week of being fed with the glucose-glycerol mixture. in the glycerol-fed reactor where a more diverse population was present, there was a microorganism (band number 57, fig. 6 ) that after only three volume changes took advantage of the presence of glucose besides glycerol. after 1 week, the bands 53, 61 and 58 almost disappeared, and another population emerged represented by the three bands that are positioned above band 57. these three bands seem to be an artefact of band 57, since only one could be successfully purified and gave the same sequence. in the bioreactor initially fed with xylose, an analogous effect was observed to the reactor fed with glycerol. one week after additional glucose was supplied, bands numbers 9 and 23 remained in the system, band 18 disappeared and band 83 emerged. in this study, a mixture of two inocula was used in order to initially provide a high microbial diversity. the high diversity is reflected in the different microbial populations established with different substrates and under the different conditions studied as indicated in the phylogenetic tree (fig. 2a,b) . a low number of significant bands was found upon feeding with a single substrate: between one and three when glucose was the substrate and less than six for glycerol and xylose. at the relatively high dilution rates applied (0.04-0.13 h −1 ), it can be safely assumed that the microorganisms identified are metabolically active and directly involved in the substrate conversion. this also means that at each operational condition, the main metabolic functions are catalysed by a limited number of species. such a result was expected based on the constant operational conditions for a long period of time (at least 30 volume changes). the chemostat-type reactor is furthermore characterised by the absence of dynamics, gradients, not allowing for niche differentiation and establishment of a mixed culture with a high metabolic diversity. several theories have been formulated to elucidate the relationship between species diversity and ecosystem functioning (lawton and brown 1994; verstraete et al. 2007) . by cultivating in a chemostat, selection occurs for those microorganisms that show a higher specific substrate affinity (μ max /ks). on the other hand, there is significant redundancy in microbial species functions. fermentation of carbohydrates is a widespread function over many different genera, and as long as all the functional groups are represented, the functioning of an ecosystem does not depend on species diversity (lawton and brown 1994) . this explains the finding of similar, but different, microorganisms at comparable operational conditions such as a low or high ph value. nevertheless, when the conditions were changed as by the increase of influent substrate fig. 5 main catabolic products on one (i) and two substrates (iii) in a chemostat at ph 8, dilution rate 0.12 h −1 and 30°c. a culture cultivated on glucose before (i) and 1 week after (iii) being fed with a mixture of glucose and glycerol; b culture cultivated on glycerol before and 1 week after being submitted to a mixture of glucose and glycerol; c culture cultivated on xylose before and being submitted to a mixture of glucose and xylose for 1 week fig. 6 dgge (30-60%) pattern of the 16s rrna gene fragments of the dna extracted from the bioreactors cultivated on different substrates before and after being submitted to mixtures of two substrates: glucose, glycerol and xylose. samples taken one day before (lane i) 1 day after changing the feeding solution (three volume changes, lane ii), 1 week after (20 volume changes, lane iii) and 2 weeks after being changed back to the initial conditions (lane iv) concentration or by the introduction of a different substrate, previously absent bands emerged, suggesting that nondominant microorganisms were still present in low numbers and took over the main metabolic processes in the bioreactor. it is noteworthy that the operation time of the xylose grown culture was at least three times longer than the glucose and glycerol cultures. nonetheless, the number of significant microorganisms is higher in the xylose-cultivated community. coexistence of more than one microorganism in chemostat has been reported when more than one nutrient limitation or different environmental factors exert the control on the growth of the different microbial groups (gottschal 1993; harder et al. 1977; powell 1958; taylor and williams 1975; yoon et al. 1977) . another aspect could be the effect that a certain species can have on the growth of other species, creating a symbiotic relation. the ph was shown to have a clear impact on both the product spectrum and the microbial community structure in a glucose fermenting system. such an impact of the environmental ph on the process that results in a shift in product spectrum has been reported before horiuchi et al. 2002; zoetemeyer et al. 1982) . product spectra similar to those obtained in this study at low and high ph have been found in other studies as well (zoetemeyer et al. 1982) . at middle ph, however, many other product combinations have been reported for similar operational conditions. these product combinations mainly vary from butyrate to propionate or ethanol and acetate horiuchi et al. 1999; horiuchi et al. 2002; walker et al. 2005) . this suggests that at around neutral ph values, the ph does not impose a strong selective pressure on the system. in these cases, other factors might become important, like an increased bicarbonate concentration that may favour propionate production (via succinate; koussemon et al. 2003; van der werf et al. 1997) . such product shift could not be explained using only bioenergetic considerations at different ph values because both conversions are equally favourable in terms of gibbs free energy change, and the estimated atp yields on substrate (y atps ) are also comparable (table 1) . a higher atp efficiency, as found at lower ph values, is justifiable due to the higher maintenance requirements: & a higher fraction of non-dissociated acids in solution, & a lower dilution rate at lower ph values (ph≤5.5), and & the higher toxicity of butyrate compared to acetate (herrero et al. 1985) . therefore, at least two metabolic properties can be distinguished with respect to acid toxicity tolerance at low and high ph values (outside the range of ph values 6-7). the presence of clostridiaceae has been widely reported in similar operational conditions: fermentation of carbohydrates, mesophilic temperatures, anaerobic conditions, dilution rate between 0.04-0.2 h −1 and ph from 4-8 iyer et al. 2004; kim et al. 2006; ren et al. 2007; ueno et al. 2001) . the cluster i of the genus clostridium are strictly anaerobic "gram-positive" microorganisms (collins et al. 1994) . little is known about the difference within this cluster in terms of acid tolerance. most clostridia cannot grow at ph values lower than 6.2 or need to change their metabolism by producing solvents instead of fatty acids (svensson 1992; wiegel et al. 2006) . here, however, growth occurred with formation of acids at low ph. the microorganisms found at low ph were closely related to c. acidisoli and c. pasteuranium, species that have been described as acid-tolerant and grow at ph 3.6-7.0 (wiegel et al. 2006) . when different substrates were tested at the same ph, different populations established. part of the metabolic pathways utilised for degradation of the different substrates is comparable, but the substrate uptake mechanisms or the substrate oxidation state may induce different pathways. as the influent substrate concentration was increased, a shift in the metabolism was observed with all the carbon sources tested. however, this functional shift was not always followed by a shift in the population (glucose and glycerol) calculations were made based on the standard free energy of formation and corrected to ph 7 (hanselmann 1991) . the yield of atp was estimated based on the catabolic products, assuming glycolysis occurs through the embden meyerhof parnas pathway (emp) and substrate level phosphorylation (slp) involved in each product formation pathway. where the same communities remained with a different metabolism. this suggests that the product shift was rather a metabolic response to the operational conditions than a different community selection. fermentation of a glucose-glycerol mixture resulted in a comparable product spectrum, independent of the fact that it has been previously cultivated on glucose or on glycerol. the response of the microbial community, however, was strongly dependent on the cultivation history. once a second substrate is added, the present population can either be able to use it or not. examples of both populations were observed during these experiments. specialised microorganisms as represented by bands with the numbers 18, 53, 58 and 61 lost some advantage by the presence of a second substrate, whereas mixotrophic microorganisms as 9, 23, 45 and 57 that could convert a second substrate had a competitive advantage under these conditions and started to dominate the microbial community. after reversing the substrate to only xylose or glycerol, the bands that emerged on the substrate mixture disappeared, and the original microbial composition was reestablished. mixotrophs have a competitive advantage over the specialists when fed with a mixture of substrates. in a chemostat, mixotrophs convert two substrates while maintaining the same growth rate equal to the dilution rate. this means that the individual growth rates on each substrate can be lower than when growing on a single substrate. as all the kinetic parameters remain the same, a lower rate is only associated to a lower substrate concentration inside the reactor. under these conditions, the substrates concentrations maintained by the mixotrophs become lower than the concentrations established by the specialist population that will be washed out from the chemostat. this phenomenon has been extensively studied and described with similar results for pure cultures with different substrates (kovarova-kovar and egli 1998; kuenen 1983; yoon et al. 1977) . at this stage, the study of the microbial composition is an interesting tool to investigate mixed culture processes. it 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explanation for the toxicity of fermentation acids at low ph-anion accumulation versus uncoupling denaturing gradient gel electrophoresis in marine microbial ecology clostridium-quinii sp-nov, a new saccharolytic anaerobic bacterium isolated from granular sludge theoretical studies on coexistence of competing species under continuous-flow conditions influence of the ph on (open) mixed culture fermentation of glucose: a chemostat study glycerol fermentation by mixed cultures, a chemostat study environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by actinobacillus sp. 130z microbial resource management: the road to go for environmental biotechnology ph and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon an introduction to the family clostridiaceae failure of the ammonia oxidation process in two pharmaceutical wastewater treatment plants is linked to shifts in the bacterial communities competition for mixed substrates by microbial-populations influence on acidogenic dissimilation of glucose in an anaerobic digester acknowledgements this work was funded by the dutch technology foundation (stw), project no. dpc5904. the authors want to thank to yang jiang and esengül yildirim for helping and performing some of the experiments reported in this article.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012099-fveq5c9w authors: chao, yuanqing; zhang, tong title: optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy date: 2011-09-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3551-5 sha: doc_id: 12099 cord_uid: fveq5c9w fixation ability of five common fixation solutions, including 2.5% glutaraldehyde, 10% formalin, 4% paraformaldehyde, methanol/acetone (1:1), and ethanol/acetic acid (3:1) were evaluated by using atomic force microscopy in the present study. three model bacteria, i.e., escherichia coli, pseudomonas putida, and bacillus subtilis were applied to observe the above fixation methods for the morphology preservation of bacterial cells and surface ultrastructures. all the fixation methods could effectively preserve cell morphology. however, for preserving bacterial surface ultrastructures, the methods applying aldehyde fixations performed much better than those using alcohols, since the alcohols could detach the surface filaments (i.e., flagella and pili) significantly. based on the quantitative and qualitative assessments, the 2.5% glutaraldehyde was proposed as a promising fixation solution both for observing morphology of both bacterial cell and surface ultrastructures, while the methonal/acetone mixture was the worst fixation solution which may obtain unreliable results. electronic supplementary material: the online version of this article (doi:10.1007/s00253-011-3551-5) contains supplementary material, which is available to authorized users. the observation of the morphology of bacterial cell and their ultrastructures is fundamental for understanding the structure and behavior of bacteria, since morphology is one way for bacteria to cope with their environment and gain a competitive advantage (young 2007) . to facilitate accurate observation, various fixation methods were widely applied to fix cells (moloney et al. 2004) . the main objectives of fixation were to inhibit cellular autolysis, to preserve cellular components and morphology, and to present cells with a distinct microscopical appearance (paavilainen et al. 2010) . fixation methods can be divided into two groups: additive and denaturing fixations (st-laurent et al. 2006) . additive fixation solutions (also called cross-linking fixations) contain various aldehydes, including formaldehyde, paraformaldehyde, glutaraldehyde, etc., and can create covalent chemical bonds between proteins (dapson 2007) . this method can preserve the natural structure of proteins, i.e., secondary and tertiary structures (meade et al. 2010) . another group is the denaturing (or precipitating) fixations. these methods can denature proteins by reducing their solubility and/or disrupting the hydrophobic interactions, and thus modify the tertiary structures of proteins as well as inactivate enzymes (st-laurent et al. 2006) . alcohols, such as methanol and ethanol, are commonly used for denaturing fixation. however, alcohols are seldom solely applied since they can induce serious cell shrinkage. other denaturing chemicals, like acetone and acetic acid, are usually combined with alcohols to enhance the fixation performance (moloney et al. 2004) . to select the promising fixation methods, a number of studies have been conducted to evaluate the performance of different methods in fixing various cells or tissues (suthipintawong et al. 1996; hoetelmans et al. 2001; moloney et al. 2004; vekemans et al. 2004; celie et al. 2005; st-laurent et al. 2006; meade et al. 2010) . unfortunately, the above studies mainly focused on the cells or tissues from animals or human. the effects of different fixation methods on bacterial morphology were rarely studied, and thus research gaps still remained in this issue. microscopical methods were commonly used in previous studies to evaluate the performance of the fixation methods, including light microscopy (st-laurent et al. 2006) , reflection contrast microscopy (hoetelmans et al. 2001) , fluorescence microscopy (celie et al. 2005) , raman microscopy (meade et al. 2010) , electron microscopy (hoetelmans et al. 2001) , as well as atomic force microscopy (moloney et al. 2004) . atomic force microscopy (afm) has been widely used in all fields of surface science since its invention in 1986, including microbiological studies (bolshakova et al. 2004 ). comparing with other traditional microscopes, one of afm's advantages is acquiring three-dimensional morphological images at nanometer or sub-nanometer scales under either dry or wet conditions (gaboriaud and dufrêne 2007) . this provides great application potentials for quantitative measurements of the morphology of bacterial cells and surface ultrastructures. a few previous studies evaluated the effects of different afm scanning modes on the cell morphology of several bacteria (camesano et al. 2000; bolshakova et al. 2001; pelling et al. 2005; arce et al. 2009 ). moreover, the bacterial ultrastructures, including flagella (touhami et al. 2006) , pili (pelling et al. 2005; touhami et al. 2006; arce et al. 2009 ), and capsules (stukalov et al. 2008) , were also observed and quantified, based on the exquisite sensitivity and high spatial resolution of afm. however, as far as we know, studies were rarely conducted to evaluate the effects of fixation methods on morphology of bacterial cells and ultrastructures by using afm. to fill the mentioned research gaps, the present study was conducted to determine the effects of different fixation methods on the morphology of bacterial cell and its ultrastructures by using afm and to evaluate the fixation ability of applied methods via both qualitative and quantitative assessments. three reference bacterial strains were used in the present study. two gram-negative bacteria, escherichia coli wildtype strain k-12 and pseudomonas putida dsm 291 type strain, were purchased from the e. coli genetic stock center (department of biology, yale university) and deutsche sammlung von mikroorganismen und zellkulturen gmbh (dsmz), respectively. gram-positive bacterium, bacillus subtilis atcc 6633, was purchased from difco laboratories (detroit, usa). the bacteria were cultivated at 37°c and 150 rpm in sterilized (121°c for 20 min) luria-bertani (lb) medium. then, the cells were harvested in the log phase at a concentration equivalent to an optical density at 600 nm (od 600 nm ) value of ∼0.1. these bacterial cells were then used for further experiments immediately. glass slide was chosen as the substratum for afm measurement in the present study. glass slides were firstly immersed in ethanol/hcl (v/v 70/1) solution overnight. after that, slides were washed by sonication for 10 min in sterilized di water. this procedure was repeated twice. then, the washed slides were placed in sterilized petri dishes and dried at room temperature for 12 h. finally, the prepared glass slides were stored in a desiccator before use. before fixation, bacterial cells were washed twice in phosphate-buffered saline (pbs, 8.475 g nacl, 1.093 g na 2 hpo 4 , and 0.276 g nah 2 po 4 in 1 l di water; ph 7.4). five common fixation methods (moloney et al. 2004; celie et al. 2005) were applied to fix the washed cells, including 2.5% glutaraldehyde in pbs for 2 h, 4% paraformaldehyde in pbs for 30 min, 10% formalin in pbs for 10 min, methanol/acetone (1:1) for 10 min, and ethanol/acetic acid (3:1) for 10 min. all fixations were conducted at room temperature. after fixation, the cells were washed twice in pbs and then re-suspended in sterilized ultrapure water to avoid salts crystallization during dry process and subsequent influence on afm measurement. finally, 100 μl of prepared bacterial solution was dripped onto the glass slide and air-dried. all the samples were stored at 4°c before afm measurement. all fixation methods were conducted with two to three duplicates. afm measurements afm images were acquired by using tapping mode of jpk nanowizard afm (jpk instruments, germany). silicon cantilever tap300 (budgetsensors, bulgaria) with a resonance frequency of 300 khz and a spring constant of 40 n/ m was applied to analyze the air-dried samples in air. the tip radius of cantilever is less than 10 nm and opening angle is between 40°and 50°according to the manufacturer. to decrease the applied force between cantilever tip and bacteria to minimize the influence to the bacterial morphology during afm scanning, the amplitude set point was maintained at a high level relative to the free amplitude of the cantilever (camesano et al. 2000) , since the applied force to sample from cantilever is negatively correlated with the amplitude value under afm tapping mode. measurements were started by scanning a random area of 50×50 μm 2 which could contain several to dozens of bacterial cells. these images were used to evaluate the morphology of bacterial cells. then, the scan size was decreased gradually until bacterial pili or flagella could be observed clearly. amplitude and phase images were recorded simultaneously with height images. height images revealed the sample topography and were applied to quantify the morphology of bacterial cells, flagella, and pili. height images were also used to calculate the roughness of bacterial surface based on root mean square (rms) values, i.e., the standard deviation of all the height values within the given area (camesano et al. 2000) . rms roughness was widely used as an important parameter to describe bacterial morphology in previous studies (auerbach et al. 2000; camesano et al. 2000; pelling et al. 2005; alsteens et al. 2008; andre et al. 2010) . the measurements were conducted over two different areas (0.5×0.5 μm 2 ) on the surface of one individual cell, and there were 10 to 20 measurement duplicates for each bacterium/fixation combination. amplitude images were captured to analyze surface features since they have higher sensitivity than height images (pelling et al. 2005) . phase images were applied to reveal the sample heterogeneity since the phase signal is sensitive to properties of the tip-sample interaction and may show overall mechanical, chemical, and topographic properties of the samples (camesano et al. 2000) . more than eight images were captured for each treatment. ten to twenty cells, flagella, and pili were taken into morphology analysis for each treatment. noticeably, due to the "side-wall" artifact ( fig. s1 ), the image width of bacterial filaments (e.g., flagella and pili) might be overestimated comparing to their true width (bolshakova et al. 2004; harada et al. 2008; kuznetsov and mcpherson 2011) . the error between the image and true width is a function of the width of the cantilever tip (kuznetsov and mcpherson 2011) . in the present study, the error was predicted and the true width of bacterial filaments was corrected to accurately evaluate the fixation ability (please refer to the supporting information for details). for bacterial cells, no correction was conducted since the "sidewall" artifact could be negligible at micron scale. as shown in fig. 1 , single e. coli (fig. 1a) and p. putida ( fig. 1b ) cells were evenly separated on the glass slide, while b. subtilis formed multicellular chains (fig. 1c) . the morphological data (table 1) showed that the fixed and unfixed cells were flattened on the glass slide since the height values of bacteria were lower than the width values. thus, to quantitatively evaluate the effect of fixation methods on bacterial morphology, the width/height ratio (w/h) was firstly used as an index to reflect the preservation of bacterial cell shape after fixation since the natural w/h of an intact cell is about one for rod-shaped bacteria. the results in table 1 showed that all the w/h values of cells after the fixation treatments were significantly lower (p<0.01) than those of no fixation treatments except for p. putida (p=0.09) and b. subtilis (p=0.87) treated by the methanol/acetone mixture. this revealed that the applied fixation methods could reduce the influence of dehydration during air-dry process and therefore effectively maintain cell morphology. among these fixation solutions, the ethanol/acetic acid solution performed the best since the w/h ratios of cells fixed by it was the closest to 1, especially for p. putida and b. subtilis. the 2.5% glutaraldehyde, 10% formalin, and 4% paraformaldehyde solutions showed their medium preservation ability, while the methanol/acetone solution had the highest w/h and therefore showed the worst performance among the five fixation methods. bacterial surface roughness, in terms of the rms value, was another quantitative index to evaluate the cell surface morphology. the results in table 1 showed that the tested bacteria had rough surfaces. for the two gram-negative strains, i.e., e. coli and p. putida, their rms roughness values fells into the similar ranges, i.e., 9.1-20 and 8.1-21 nm, respectively. however, the roughness of grampositive strain b. subtilis was in a higher range varying from 18 to 40 nm. several cellular ultrastructures were detected from afm images, including flagella, pili, and extracellular polymeric substance (eps). to eliminate the "side-wall" artifacts, the corrected widths for bacterial filaments (i.e., flagella and pili) were calculated according to the image width and tip geometry of applied cantilever (please refer to supporting information for details). multiple flagella could be observed for all the tested bacteria, having a width of 40-90 nm and a height of 8-12 nm ( table 2 ). the peritrichous e. coli and b. subtilis had flagella all round the cell while the flagella of p. putida were only found at the cell pole. the flagella of bacterial cells fixed by 2.5% glutaraldehyde and 10% formalin were preserved better than those without fixation (fig. 2) . the flagella could also be detected in the treatment using 4% paraformaldehyde. however, the amount of flagella per cell decreased significantly and many of them were detached from the cell body or had incomplete structure (white arrows in fig. 2 a-d and c-d). for the methanol/acetone and ethanol/acetic acid groups, the flagella totally disappeared. pili are another filamentous structure and only detected from e. coli. there were several distinct differences between pili and flagella in afm images. first, the size of pili was significantly smaller than flagella with corrected widths of 17-33 nm and heights of 4.3-6.2 nm, respectively (table 2) . second, the pili had linear structure while flagella were helical filaments. third, the length of pili was significantly shorter than that of flagella (figs. 2 and 3 (table 3) . for the treatment using 10% formalin, most of pili were also detached from the cell body (fig. 3c) . however, the detached pili still remained on the substrata surface, and thus their morphology could be measured. the 2.5% glutaraldehyde solution showed the best performance since the pili were intact and maintained in the natural morphology. eps could also be observed in the phase images since its physicochemical and topographic properties were different from bacterial cells (fig. 4) . the eps of e. coli and b. subtilis was observed in several fixation treatments, including 10% formalin, 4% paraformaldehyde, and the methanol/ acetone solutions (figs. 1, 2, and 4) . this indicated other fixation methods using 2.5% glutaraldehyde, and the ethanol/acetic acid solution significantly removed the eps. this is a good point for observing the morphology of bacterial cells and surface ultrastructures since (1) the eps might interfere the cell edge estimation and consequently affect the morphology observation, (2) other ultrastructures like flagella and pili might be covered by the dehydrated eps and thus could not be observed in afm images, and (3) eps might contaminate the cantilever tip during scanning and finally deteriorate the image quality. debris and coating artifacts caused by the fixation two types of artifacts, i.e., debris and coating, caused by the fixation, could occur in the afm images. the debris artifacts on the sample surface, which mainly came from disintegration of bacterial cells during washing and fixation processes, generated false morphological data and thus affected the morphology analysis significantly (moloney et al. 2004) . in afm images, the visible debris artifacts were in smaller size than bacterial cells, had heterogeneous morphology, and commonly existed on the sample surface ( figs. 1 and 2) . based on the amount and size of debris, the ethanol/acetic acid solution seemed to be the best fixation method generating little debris. debris with larger amount and size were found in the samples fixed by 10% formalin and the methanol/acetone solution, indicating that these two fixation methods were less preferred due to debris generation and the unreliable morphology. coating artifacts were a continuous layer composed of bacteria-derived materials and/or residual culture medium which deposited on the sample surface (moloney et al. 2004) . coating artifacts might mask the true topography of bacterial surface and could also significantly interfere the bacterial morphology determination. moreover, the surface ultrastructures, such as flagella and pili, might be embedded in this thin layer and not be able to be detected totally. therefore, the coating effect should be taken into consideration when choosing the fixation methods. the results of the present coli which were air-dried only (a) and fixed by 2.5% glutaraldehyde (b), 10% formalin (c), 4% paraformaldehyde (d), methanol/acetone (e), and ethanol/acetic acid (f). the white arrow showed the surrounding eps. the black arrows in c indicated the coating artifacts study showed the coating artifacts appeared in the afm images of several fixation methods, especially 10% formalin (black arrows in figs. 2, 3, and 4 ). this revealed that the afm images of samples fixed by 10% formalin might face the problem of coating artifacts. several studies had been conducted to determine the cell morphology of various bacterial species using different afm operational modes either in air or under aqueous conditions ( table 4 ). the observed bacteria were not natural rod-or sphere-shape, but flattened to certain extents. several factors, including environmental variables as well as afm operations, could significantly interfere the morphology determination (bolshakova et al. 2004 ). the scanning conditions were critical for acquiring the bacterial morphology. typically, bacterial morphology in air was flatter than those in liquid (table 4) , since the dehydration of cell and surface eps in air might mainly affect the measurement. another factor was afm scanning mode, i. e., contact and tapping modes. for the contact mode, the cantilever tip was always touching with the bacterial cells under a given force during scanning. although the selected cantilever for the contact mode was relative soft, the interaction between the cantilever tip and the cells could significantly interfere the cell morphology observation, such as bacterial distortion or detachment caused by the lateral force as well as artifacts caused by the cantilever contamination, especially for the soft cell and deformable eps. for the tapping mode, the contact between the cantilever tip and cell surface only lasts for a small portion of oscillation cycle, i.e., about at the lowest point of an oscillation cycle. this scanning mode could significantly decrease the sample damage and lateral force (camesano et al. 2000) . in the present study, the relatively "light" tapping mode by applying larger oscillation amplitude further reduced the influence of interaction between the cantilever tip and cells on cell morphology determination. the bacterial filamentous structures, such as flagellum and pili, were observed using afm in a few previous studies (pelling et al. 2005; touhami et al. 2006; arce et al. 2009 ). unlike bacterial cells, the filamentous structures were difficult to be detected in aqueous environments via afm since the force to detach bacterial filaments from the substrata was relatively small, and thus these structures were easily detached in a fluid medium (touhami et al. 2006 ). however, it was easier to detect these filamentous structures after drying and by scanning in air since the adhesion strength increased significantly after drying (roosjen et al. 2005 ). in the present study, the flagella of three tested bacteria and pili of e. coli sahu et al. 2009 a for rod-shaped bacteria, the data were widths values; for spherical bacteria, the data were diameter b the data were collected from figures of cross-section profile in reference were clearly observed in air, and their morphologies were determined accordingly by analyzing afm height images. the dimension of bacterial flagella in the present study varied between 40 and 90 nm in width (the "side-wall" artifact was taken into consideration) and between 8 and 12 nm in height (table 2) and was compatible with the flagellar sizes of others' studies, which had a width of 17-48 nm and a height of 5.5-6.5 nm (jaschke et al. 1994) , as well as a width of 50-150 nm and a height of ∼4 nm (schmid et al. 2008) . assuming that flagella were deformable and the cross section was ellipse, the perimeter of the ellipse was equal to that of circular cross section of natural and undeformed flagella (i.e., l ¼ ph þ 2ðw à hþ ¼ pd). from the corrected data of flagellar width and height, the converted diameters of the three tested bacteria could be calculated (d ¼ h þ 2ðw à hþ=p). the converted diameters varied from 14 to 28 nm for e. coli, 17 to 24 nm for p. putida, and 17 to 23 nm for b. subtilis. this was the typical dimension of bacterial flagella within the diameter of 12 to 25 nm (namba and vonderviszt 1997; samatey et al. 2001; maki-yonekura et al. 2010) . for e. coli pili, the dimension observed in the present study had a corrected width of 17 to 33 nm, a height of 4 to 6 nm, and a converted diameter of 6 to 10 nm. this was also compatible with the typical dimension of bacterial pili, which had 4 to 7 nm diameter according to previous reports (korhonen et al. 1980; telford et al. 2006; touhami et al. 2006; arce et al. 2009 ). to optimize the fixation methods for the morphology observation of bacterial cells and surface ultrastructures, five common fixation solutions were evaluated on their preservation ability in the present study. the results strongly suggested that the fixation methods could significantly affect the morphology of bacterial cell as well as the surface ultrastructures. the fixation methods containing alcohols (including the methanol/acetone and ethanol/acetic acid solutions) obtained biased morphology than those containing aldehydes (including 2.5% glutaraldehyde, 10% formalin, and 4% paraformaldehyde), since the filamentous structures (flagella and pili) disappeared on the cell surface. this might be mainly caused by the alcohols in these fixation solutions which could dissolve the membrane lipids, form large pores in the cell, and detach the surface macromolecules on the cell surface (vekemans et al. 2004 ). for the cell morphology, the ethanol/acetic acid solution could obtain better preservation than the methanol/acetone solution (table 1 and fig. 1 ). in the ethanol/acetic acid combination, acetic acid could swell cell and thus counteracted the shrinkage induced by ethanol, while the main role of acetone in the methanol/acetone solution was penetrating cell to facilitate the consequent fixation by methanol (st-laurent et al. 2006 ). thus, the bacteria cell fixed by the methanol/acetone solution shrank seriously, and the bacterial morphology was poorly preserved. other studies also revealed that no reliable morphology could be obtained after acetone/methanol fixation (hoetelmans et al. 2001; st-laurent et al. 2006) . the fixation methods applying aldehydes showed medium preservation ability for cell morphology judging from the w/h ratios (table 1) . for bacterial filaments morphology, the aldehyde fixations performed much better than alcohols since aldehydes fixed cell by forming covalent chemical bonds between proteins and therefore could maintain the integrality of membrane lipids as well as the surface macromolecules (dapson 2007 ). in the present study, 2.5% glutaraldehyde showed the best performance for fixation of filaments, following by 10% formalin and 4% paraformaldehyde. paraformaldehyde, the polymerized form of formaldehyde, would be depolymerized to formaldehyde when dissolved (kiernan 2000) . therefore, the 4% paraformaldehyde solution contained pure formaldehyde. for the 10% formalin solution, the major component is formaldehyde but also contains 1% methanol which is added to slow down the polymerization of formaldehyde (kiernan 2000) . this might be the main reason to explain the slightly different performances between 10% formalin and 4% paraformaldehyde (tables 1 and 2) . comparing with formaldehyde, glutaraldehyde could fix sample more tightly since it has longer molecule and two aldehyde groups which has potential to link more distant protein molecules (kiernan 2000) . this might explain the best performance of glutaraldehyde in fixing the bacterial filaments among the applied fixation methods. the bacterial surfaces were not smooth but with lots of spherical structures (phase images in figs. 3 and 4) . these structures were always observed in the images of different bacteria fixed with aldehydes, indicating they were the bacterial morphological feature, instead of artifacts formed in drying and fixation or due to the cantilever contamination. these spherical structures were also observed in a few previous studies and considered to be the surface proteins (camesano et al. 2000; micic et al. 2004 ) and/or lipopolysaccharides (micic et al. 2004; handa et al. 2010; mcewen et al. 2010) . comparing with aldehyde fixations, the bacteria fixed by alcohols showed obscure spherical structures (fig. 4) . this further indicated that the alcohols could remove the surface proteins and/or lipopolysaccharides. to comprehensively evaluate the applied fixation methods and determine which one was the most promising method, quantitative and qualitative assessments were conducted in the present study (table 5) . quantitative assessments were based on the morphology preservation of both bacterial cell and surface filaments by comparing the w/h ratios of cells fixed using different methods. qualitative assessments were based on the eps removal, debris, and coating artifacts by analyzing afm images. other possible artifacts, such as salt crystals and streaking artifacts (moloney et al. 2004) , could also affect the morphological analysis. salt crystals might derive from washing buffer (pbs or others) after drying. to avoid crystals generation, the ultrapure water was applied to re-suspend the fixed cells before drying in air. the afm images also showed no salt crystals generated on the sample surface. streaking artifacts are mainly caused by the inappropriate afm operation or tip contamination which might lead into inaccurate measurements and consequently interfere the morphology analysis, but having nothing to do with the fixation methods. in the present study, the operation parameters such as set points and scan rates were optimized in the pre-experiments and also adjusted during scanning to avoid the streaking artifacts due to inappropriate afm operations. for bacterial samples, the tip contamination was almost inevitable since the cell or debris which adhered loosely on the cell surface easily adhered to the tip during scanning. the strategy applied in the present study was to replace the contaminated cantilever immediately with a new one when the streaking artifacts appeared in an image. therefore, the salt crystals and streaking artifacts were not taken into consideration for the assessments of the fixation methods. based on above assessments, several comments were also proposed in table 5 for future applications. organization of the mycobacterial cell wall: a nanoscale view imaging the nanoscale 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and source are credited. key: cord-012054-bpgb7tgo authors: ferreira, maria isabel m.; marchesi, julian r.; janssen, dick b. title: degradation of 4-fluorophenol by arthrobacter sp. strain if1 date: 2008-03-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1343-3 sha: doc_id: 12054 cord_uid: bpgb7tgo a gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. the organism, designated strain if1, was identified as a member of the genus arthrobacter on the basis of 16s ribosomal rna gene sequence analysis. arthrobacter strain if1 was able to mineralize 4-fp up to concentrations of 5 mm in batch culture. stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-fp metabolism. the degradative pathway of 4-fp was investigated using enzyme assays and identification of intermediates by gas chromatography (gc), gc–mass spectrometry (ms), high-performance liquid chromatography, and liquid chromatography–ms. cell-free extracts of 4-fp-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. cells grown on 4-fp oxidized 4-fp, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. during 4-fp metabolism, hydroquinone accumulated as a product. hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and β-ketoadipic acid. these results indicate that the biodegradation of 4-fp starts with a 4-fp monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the β-ketoadipic acid pathway. during the past decades, widespread application of fluoroaromatic compounds as agrochemicals and pharmaceuticals has lead to an increased occurrence of environmental contaminants containing fluorine (key et al. 1997) . fluorinated compounds are rare in nature (harper and o'hagan 1999) . the stability of the carbon-fluorine bond (116 kcal/mol in ch 3 f, compared to 81 kcal/mol for the carbon-chlorine bond in ch 3 cl) makes most fluorinecontaining compounds much more resistant to biodegradation than their unsubstituted analogs (key et al. 1997) . furthermore, the van der waals radius of fluorine is small (1.47 å, in between that of a hydrogen and an oxygen). yet, the electronegativity of fluorine causes strong polarization of c-f bonds, and fluorine substituents may be involved in biological interactions (howard et al. 1996) . regardless of the recalcitrance of most fluoroaromatics to biodegradation, several bacterial cultures have been described that aerobically degrade fluorobenzoic acids (oltmanns et al. 1989; engesser et al. 1980; harper and blakley 1971; schlomann et al. 1990 ). research on bacterial fluorophenol degradation has been limited to studies with whole cells, cell extracts, or purified enzymes from rhodococcus species that were obtained by enrichment with other aromatic compounds as a growth substrate (boersma et al. , 2001 bondar et al. 1998 bondar et al. , 1999 finkelstein et al. 2000) . the fluorobenzene-degrading organism rhizobiales f11 could grow on 4-fluorophenol (4-fp), but information on the pathway of 4-fp metabolism is lacking. cometabolic degradation of difluorophenols and trifluorophenols by several rhodococcus species is initiated by a phenol hydroxylase that catalyzes ortho-hydroxylation, resulting in the formation of the respective fluorocatechol, which is then cleaved by an intradiol dioxygenase to produce fluoromuconate (bondar et al. 1998 ). conversion of 4-fp by whole cells of the phenol-degrading organism rhodococcus opacus 1cp resulted in the formation of 4fluorocatechol, 1,2,3-trihydroxy-5-fluorobenzene, and fluoromuconates (finkelstein et al. 2000) . yeasts and fungi that are able to cometabolically transform fluorinated phenols have also been described. whole cells of exophiala jeanselmei transformed 4-fp into 4-fluorocatechol and 3fluoromuconate . penicillium frequentans metabolized monofluorophenols in the presence of glucose or phenol. the metabolism of meta-or parafluorophenols yielded the corresponding catechol and 4-carboxymethylenebut-2-en-4-olide (hofrichter and schreibner 1993; hofrichter et al. 1994) . none of these organisms could utilize fluorophenols as a growth substrate. to our knowledge, studies on the metabolism of fluorophenols by a bacterial culture that is capable of using such as compound as a sole source of carbon and energy have not been reported. in the present paper, we describe the isolation and characterization of a bacterial strain growing on 4-fp as the sole source of carbon and energy. based on the identification of several intermediates, a metabolic route for the degradation of 4-fp by this strain is proposed. media and growth conditions nb medium contained 8 g of nutrient broth (difco) per liter. mineral salts medium (mm) contained per liter 5.3 g na 2 hpo 4 12h 2 o, 1.4 g kh 2 po 4 , 0.2 g mgso 4 7h 2 o, 0.5 g (nh 4 ) 2 so 4 , 5 ml trace metals solution (janssen et al. 1985) , and 10 mg of yeast extract (difco laboratories). when required, the solid medium was obtained by adding 15 g/l of difco agar. strain if1 was grown at 30°c on a rotary shaker (180 rpm). cultures were grown in 100-ml flasks filled to 25% of their volume and were closed with teflon-lined screw caps. e. coli cells were grown in luria-bertani medium (lb) at 37°c on a rotary shaker. enrichment and isolation of 4-fp-degrading cultures a variety of soil samples, collected from different sites in the netherlands that are contaminated with halogenated aliphatic compounds (such as monochlorobenzene, hexachlor-obenzene, and trichloropropane), were used as the initial inoculum for the 4-fp enrichments. the soil samples were used to inoculate flasks containing 30 ml of sterile minimal salts medium and 1 mm of 4-fp, supplied in the liquid phase as the sole carbon and energy source. the cultures were incubated at room temperature on a rotary shaker (150 rpm), and 40% of the suspension was transferred to a new flask containing fresh medium every 15 days. during this time, growth (optical density at 600 nm) and liberation of fluoride were monitored. samples of the enrichment culture were periodically spread onto minimal salts agar plates containing 1 mm 4-fp and onto nb plates as soon as growth on 4-fp was established. pure cultures were obtained by repetitive streaking onto solid mm containing 4-fp and tested separately for growth on 1 mm 4-fp liquid medium. growth and fluoride release were again monitored to verify 4-fp degradation. strains capable of 4-fp degradation as a sole source of carbon and energy were used for further experiments. strain if1 was deposited at centraalbureau voor schimmelcultures, utrecht, the netherlands, under accession number nccb 100218. sequencing of the 16s rrna gene for cloning of the 16s ribosomal ribonucleic acid (rrna) gene, a single colony of strain if1 was directly used for polymerase chain reaction (pcr) amplification. the primers 63f (5′-caggcctaac acatgcaagtc-3′) and 1387r (5′-gggcggwgtgta caaggc-3′; marchesi et al. 1998) were used for pcr amplification. the pcr reaction mixture (50 μl) contained taq pcr buffer, 2.5 mm mgcl 2 , 20 pmol of each appropriate primer, 200 mm of each deoxyribonucleotide triphosphate, 1 u taq dna polymerase, and biomass of strain if1. the pcr conditions were 94°c for 10 min followed by 1 min at 95°c, 1 min at 55°c, 1.5 min at 72°c, and 5 min at 72°c. the resulting fragments were cloned into the pcr4-topo vector (invitrogen, carlsbad, ca) and transformed into e. coli top10 cells. the transformed cells were plated on lb plates containing 0.5 mg/ml of ampicillin, and the positive colonies were used for plasmid isolation and sequencing. phylogenetic analysis alignments of the 16s rrna gene were made using sequences downloaded from the ribosomal database project ii (rdp ii; cole et al. 2005) , after searching for nearest neighbors using the sequence match tool. further searches were conducted using blast and fasta of the european molecular biology laboratory (embl) database for 16s rrna gene sequences that are closely related to the 16s rrna gene of strain if1 but not available from the rdp ii. alignments were subsequently made using the profile alignment option in clustalx (thompson et al. 1997) , refined using bioedit ver 7.0.5.2 (hall 1999) , and subsequently parsed through gblock (castresana 2000) to remove ambiguously aligned sections and increase the robustness of the data. phylogenetic trees were determined using the neighbor-joining (nj) method. evolutionary distances for the global tree were calculated using the kimura-2-parameter model with a transition/ transversion ratio of 2 ( fig. 1) . further trees were constructed in phylip version 3.6a3 (j. felsenstein), using maximum parsimony and maximum likelihood methods. all nj trees were tested statistically by means of bootstrap analysis. preparation of cell extracts cells were grown in mm with 4 mm 4-fp, harvested by centrifugation at 5,000×g for 15 min, washed with ice-cold td buffer (0.1 m tris-hcl, ph 7.5, and 0.1 m 1,4-dithiothreitol), and stored at −20°c until further use. the frozen cells were thawed, resuspended in td buffer, and incubated with lysozyme (50 mg/ml) for 1 h at 30°c. a french press was used to disrupt the cells, and the crude extracts were centrifuged at 10,000×g for 15 min to separate the soluble from the particulate fraction. the supernatant was used for further experiments. protein concentrations were determined with the biorad protein assay kit. enzyme assays catechol 1,2-dioxygenase and catechol 2,3dioxygenase activities were measured spectrophotometrically at 260 and 375 nm, respectively. the reaction mixtures contained 0.1 mm catechol, td buffer, and cellfree extract (0.1 mg of protein) in a final volume of 1 ml. 4-fluorocatechol 1,2-dioxygenase was measured as catechol 1,2-dioxygenase but with 4-fluorocatechol instead of catechol as the substrate. the 4-fp monooxygenase activity was measured spectrophotometrically by following the consumption of nadh at 340 nm in a reaction mixture containing cellfree extract (0.1 mg protein), 0.1 mm 4-fp, 0.1 mm nadh, and buffer in a total volume of 1 ml. the observed rates were corrected for substrate-independent nadh oxidation. hydroxymuconic semialdehyde dehydrogenase was measured at 375 nm. reaction mixtures contained (in a final volume of 1 ml) about 0.05 mm freshly prepared hydroxymuconic semialdehyde, td buffer, 0.1 mm nad, and cell-free extract (0.1 mg of protein). hydroxymuconic semialdehyde was obtained by incubation of catechol with the cell-free extract of pseudomonas putida mt-2 as described previously (mars et al. 1998 hydroquinone dioxygenase was assayed spectrophotometrically by monitoring the change in absorbance between 230 and 330 nm in a reaction mixture of 1 ml final volume containing 0.1 mm hydroquinone, td buffer, and cell-free extract (0.1 mg of protein). hydroquinone hydroxylase and hydroxyquinol dioxygenase were assayed by using a fiber optic oxygen sensor. reaction mixtures contained 1 mm of substrate, mm, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml. oxygen uptake measurements strain if1 was grown with glucose or 4-fp as the sole carbon source and harvested by centrifugation at 6,000×g for 15 min at 4°c. cells were resuspended in mm, and o 2 consumption was measured with a fiber optic oxygen sensor (mops-1, prosense bv, hannover, germany). all reactions were performed in a stirred vessel at room temperature. the reaction mixtures contained 1 mm of substrate (4-fp, hydroquinone, 4fluorocatechol, hydroxyquinol, or catechol), mm, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml. analytical methods for capillary gas chromatography (gc), 4 ml samples were extracted with 1 ml of diethyl ether. a model 6890 gas chromatograph (hewlett-packard) equipped with a flame ionization detector and a hp-5 column (agilent 19091j-413, 30 m×0.25 mm×0.25 μm) were used for the analysis. gc-mass spectrometry (ms) analysis was carried out with a model 5973 mass selective detector (hewlett-packard) coupled to a hp 6890 series injector and a hp1 column (agilent 19091z-433; 30 m× 0.25 mm×0.25 μm). high-performance liquid chromatography (hplc) was carried out using a chrompack c18 column (10 cm×5 mm) connected to a jasco uv-1575 detector, which monitored absorbance at 214 and 254 nm, and operated with jasco pu-980 pumps and a jasco as-1555 sampler. the mobile phase was water/acetonitrile (70:30), 5 mm potassium phosphate (ph=3), and 340 mg/l sodium dodecyl sulfate, and the flow rate was 1 ml/min. liquid chromatography (lc)-ms was carried out with a zmd micromass spectrometer, equipped with a xterra ms, symmetry shield c8 column (4.6×150 mm), a waters 996 photodiode array detector, and a waters 2690 separations module. samples of 20 μl were analyzed, and compounds were isocratically eluted at a flow rate of 1 ml/min with a solution of water/acetonitrile (80:20) and 10 mm formic acid. concentrations of free fluoride in the culture supernatants were measured with a fluoride electrode (model 96-09, thermo russell, scotland). fresh sodium fluoride standards were prepared for calibration curves. chemicals 4-fp (>98%) was obtained from sigma-aldrich (steinheim, germany). all chemicals were of the highest purity grade available (sigma-aldrich; acros organics, geel, belgium). the compound 4-fluorocatechol was kindly provided by dr. erik de vries. purity of 4-fp, 4fluorocatechol, and hydroquinone was checked by hplc. nucleotide sequence accession numbers the 16s rrna sequence of strain if1 was deposited at genbank with the accession no. dq425093. isolation of a 4-fp-degrading bacterium to obtain a bacterial culture that is able to use 4-fp as carbon and energy source for growth, enrichments were performed and followed over time. two months of selective enrichment by repeated transfer of samples from a culture that displayed growth on 4-fp to fresh 4-fp containing media resulted in a microbial consortium that was capable of growth on 4-fp as a sole source of carbon and energy. samples of the consortium were repeatedly plated onto nb agar plates and mm agar plates containing 4-fp. this procedure resulted in the isolation of three pure strains named if1, if2, and if3. the strains were restreaked on mm supplemented with 4-fp and inoculated in liquid cultures with 1 mm 4-fp. fluoride liberation was observed for all three strains but not in control incubations to which no bacterial inoculum was added. all the three strains showed growth, as monitored at 600 nm, and thus were able to use 4-fp as a sole carbon and energy source. after 5 days of incubation in liquid culture supplied with 1 mm 4-fp, strain if1 reached 100% fluoride release and an optical density (od) of 0.119 at 600 nm. strain if2 released 76% of the fluorine and reached an od 600 of 0.09, while strain if3 reached an od of 0.076 and released 54% of the theoretical amount of fluoride ions. when a mixed culture of the three strains in liquid media was used, an od 600 of 0.217 and 100% fluorine release were measured after 4 days. apparently, the mixed culture contained, even after prolonged adaptation, organisms with varying efficiencies of 4-fp utilization. because strain if1 showed the highest degradation rates combined with stoichiometric release of fluoride, this organism was chosen for further studies. microbiological characterization strain if1 is a grampositive motile bacterium with a rhodococcus lifecycle. the optimal temperature for growth is 30°c. the 16s rrna gene sequence was determined. initial searches against the rdp ii and embl rrna databases resulted in very close associations with members of the genus arthrobacter, the closest of which were the quinaldine-degrading strain arthrobacter sp. ka1-1 (overhage et al. 2005 ) and the 4-nitroguaiacol-degrading actinobacterium arthrobacter nitroguajacolicus sp. nov (kotouckova et al. 2004 ). the 16s rrna gene sequences of these organisms were greater than 99% identical to if1's. the phylogenetic analysis places the isolates in the phylum actinobacteria and the genus arthrobacter (fig. 1) . the subtree that were obtained shows the detailed relationship of isolate if1 to other members of the genus arthrobacter. the topologies of all the trees that were obtained during statistical analysis were in agreement and clearly placed this isolate in the genus arthrobacter (data not shown). furthermore, the change in form during the growth cycle between rod and coccus is typical of the lifecycle of the arthrobacter genus. catabolic activities of arthrobacter sp. strain if1 to study the degradation potential of strain if1, a range of organic compounds were tested as growth substrates. cells were inoculated into mm, and different organic substrates were added at a concentration of 1 mm. after 72-h incubation growth, substrate disappearance and halide release were measured. growth and substrate removal were found when catechol, hydroquinone, hydroxyquinol, benzoate, phenol, 4-fluorocinnamic acid, and 4-nitrophenol were used as substrates. the organism did not grow on 2-fluorophenol, 3fluorophenol, 4-chlorophenol, 4-bromophenol, 4-iodophenol, fluoroacetate, trifluoroacetate, fluoroacetamide, trifluoroethanol, or on 2-bromoethanol. the fact that strain if1 is capable of growth on catechol, hydroquinone, and hydroxyquinol but not on 4-fluorocatechol indicates that 4-fluorocatechol is not the most likely intermediate in the 4-fp pathway, although toxic effects could also play a role. to test the range of 4-fp concentrations tolerated by strain if1, experiments were conducted in sealed flasks with 4-fp at concentrations of 1 to 7 mm as a sole carbon and energy source. control assays without 4-fp showed no growth or release of fluoride, and sterile controls showed no abiotic loss of 4-fp. between 1 and 4 mm 4-fp, the substrate was completely consumed, stoichiometric release of fluoride was observed, and biomass increased linearly with the amount of 4-fp added (fig. 2a) . this indicates that the degradation of 4-fp by strain if1 does not give large amounts of fluorinated dead-end products. concentrations of 4-fp above 4 mm caused a toxic effect on the growth of if1 (fig. 2b) . the use of 5 mm 4-fp promoted growth, but a longer lag time was observed. at 7 mm 4-fp, no growth occurred even after 15 days of incubation. growth on 4-fp and formation of metabolites a batch culture of strain if1 supplied with 1 mm 4-fp as the only source of carbon and energy was monitored in time. growth was accompanied by an increase in biomass, decrease in 4-fp, and formation of fluoride (fig. 3) . after 120 h, there was complete conversion of 1 mm 4-fp, and 1 mm fluoride was formed, indicating that there was no transient accumulation of fluorinated intermediates over the whole growth period. when cells grown on 4-fp were incubated in the presence of the iron chelator 2,2′-dipyridyl, no 4-fp degradation occurred, and no fluoride was released in the medium. this indicates that initial or further enzymes involved in 4-fp metabolism require ferrous ions for activity. to isolate intermediates of the degradation of 4-fp, samples from a batch culture containing 4 mm 4-fp were taken at appropriate intervals and analyzed by gc, hplc, and lc-ms. metabolites that were detected were numbered in order of time of appearance in the culture. gc analysis indicated that at least four metabolites were formed and degraded over time (table 1) . metabolite i, the earliest product that was observed, had the same retention time as an authentic hydroquinone standard and metabolite ii had the same retention time as a standard of hydroxyquinol. gc-ms analysis showed that the mass spectrum of metabolite i was indeed similar to that of the hydroquinone standard, with a molecular ion peak at 110 and at 55 m/z. metabolite ii gave a molecular ion peak at 126 and 63 m/z, which is typical for hydroxyquinol, and the spectrum coincided with that of a standard. metabolites v and vi were detected by gc but did not ionize in gc-ms under the conditions tested and were not identified. when the supernatant of if1 cells growing on 4 mm 4-fp were subjected to hplc analysis, four peaks were detected (table 1) . metabolite i had the same retention time as the hydroquinone standard, and metabolite ii coeluted with an authentic standard of hydroxyquinol. hydroquinone was detected in culture supernatants by hplc between 7.5 and 51 h (fig. 4) . lc-ms analysis of the extracted samples of the supernatant revealed the presence of a compound (vii) of which the negative ion spectrum shows a molecular ion peak at 159 m/z and a peak at 80 m/z, which are expected for the negative ionization of 3-oxoadipate. the above results suggest that 4-fp is initially converted to hydroquinone. the most likely enzyme involved in such a conversion is a 4-fp monooxygenase. enzyme activitiesto test inducibility of enzymes involved in 4-fp degradation, cells of arthrobacter sp. strain if1 were grown on glucose or 4-fp, washed, and tested for oxygen consumption in the presence of different substrates. washed suspensions of cells grown on 4-fp rapidly oxidized 4-fp, hydroquinone, and 1,2,4-benzenetriol (hydroxyquinol) without a lag. catechol and 4-fluorocatechol did not stimulate (table 2 ). this result indicates that 4fluorocatechol and catechol are not likely intermediates and that hydroquinone and hydroxyquinol are. glucose-grown cells did not oxidize any of the aromatic substrates tested with the exception of hydroxyquinol. this observation implies that hydroxyquinol may be converted by a constitutive oxygenase, whereas most other enzymes seem inducible. cells grown with hydroxyquinol showed complete conversion of benzenetriol in the presence or absence of 2,2′dipyridyl, indicating that the putative hydroxyquinol oxygenase does not require ferrous ions to be active and that oxidation beyond this compound is not inhibited by the chelator. the formation of hydroquinone, which can be readily oxidized by 4-fp-grown cells, is in agreement with the involvement in 4-fp degradation of a 4-fp monooxygenase that is induced by 4-fp. to study in more detail the degradation pathway of 4-fp by strain if1, cell-free extracts of if1 grown on 4-fp were investigated for the presence of several enzyme activities (table 3) . when catechol 1,2-and 2,3-dioxygenases were tested for, no activity was found. furthermore, no activity was detectable for 4-fluorocatechol dioxygenase. consequently, the pathway does not proceed through catechol or a substituted catechol. when cell extracts were assayed for 4-fp monooxygenase, activity could be found in the presence of nadh but not in the presence of nadph as the reducing cosubstrate. this observation indicates that the cells contain an nadh-dependent 4-fp monooxygenase activity. hplc assays of incubations of cell extracts with 4-fp showed that substrate conversion was complete. the formation of the putative 4-fp monooxygenase was induced by 4-fp because no activity was found in extracts of cells grown in the presence of glucose. when hydroquinone was used as an assay substrate, its typical spectrophotometric peak at 288 nm was not replaced by a peak absorbing at 290 to 320 nm, which would have pointed to formation of hydroxymuconic semialdehyde. thus, no hydroquinone dioxygenase was induced, or its product was rapidly further converted. the latter was ruled out by the observation that added hydroxymuconic semialdehyde was not converted by cell extracts of strain if1, indicating that no hydroxymuconic semialdehyde dehydrogenase was induced during growth in the presence of 4-fp. these results make it unlikely that hydroxymuconic semialdehyde is an intermediate in the 4-fp pathway by strain if1. when cell extracts were assayed for hydroxyquinol degradation, complete conversion of the substrate was observed with hplc. when the reaction was monitored spectrophotometrically, the peak at 287 nm, typical of hydroxyquinol, was substituted by a peak at 245 nm, typical of maleylacetate. this indicates the involvement of a hydroxyquinol oxygenase that converts hydroxyquinol into maleylacetate, which is further converted into 3-oxoadipate. this study reports the isolation and characterization of arthrobacter strain if1, an organism capable of growth with 4-fp as a sole source of carbon and energy. the biodegradation of 4-fp by strain if1 was analyzed by gc, gc-ms, hplc, lc-ms, oxygen uptake experiments, and measurements of enzymatic activities. a metabolic pathway is proposed on the basis of the results of this study (steps 1 and 2, ortho-cleavage) and by analogy with other systems 5) . we suggest that the degradation of 4-fp in strain if1 starts with the conversion by a monooxygenase to benzoquinone, which is immediately reduced to hydroquinone. hydroquinone then undergoes a further hydroxylation to form hydroxyquinol. this benzenetriol is the ring fission substrate, which is transformed by ortho-cleavage and yields maleylacetate, which is further converted to 3oxoadipate. up to now, two main metabolic routes for the aerobic degradation of halogenated phenols have been described in the literature. in bacteria that degrade mono-and dichlorophenols, a degradation pathway is usually observed in which the substituted phenol is hydroxylated to the corresponding catechol, which is followed by orthocleavage of the aromatic ring (haggblom 1992; hollender et al. 1997; wieser et al. 1994 ). on the other hand, biodegradation pathways in which the substituted phenol is converted via hydroquinone to maleylacetate have been found, mainly in organisms that grow on polyhalophenols or 4-nitrophenol (kiyohara et al. 1992; xun et al. 1992; kadiyala and spain 1998; nordin et al. 2005; perry and zylstra 2007) . most studies have focused on the organisms and pathways that involve degradation via catechols. a clear indication for the first step in the 4-fp catabolic pathway of strain if1 was obtained by mass spectroscopic analysis of culture supernatants, which indicated the formation of hydroquinone as an early nonfluorinated intermediate. this result, in combination with the enzyme assays and oxygen uptake experiments, suggests the involvement of an inducible 4-fp monooxygenase that is nadh dependent, although the first expected product of this reaction, i.e., benzoquinone, was not detected. monooxygenation of an aromatic substrate carrying an electronwithdrawing halogen group or a nitro-substituent can result in simultaneous hydroxylation and dehalogenation or nitrite removal. this has, for example, been described for the conversion of tetrafluoro-p-hydroxybenzoate by parahydroxybenzoate hydroxylase (husain et al. 1980 ) and the oxidation of 2,4-6-trifluorophenol by a monooxygenase from ralstonia eutropha jmp134 (xun and webster 2004) . the quinone that is produced is chemically reduced by nadh to a hydroxyquinol, leading to a net stoichiometry of two nadh oxidized per halide or nitrite that is released (fig. 5) . as in our case, no transient accumulation of a benzoquinone is usually observed, indicating that benzoquinone reduction is rapid. other examples of halophenol degradation with formation of hydroquinone derivatives are the degradation of trichlorophenol to 2,6-dichlorohydroquinone by a strain of pseudomonas pickettii (kiyohara et al. 1992) , the degradation of pentachlorophenol by a sphingobium sp. through hydroxylation by a flavoprotein monooxygenase (xun and orser 1991) , and the degradation of 4-chlorophenol by an arthrobacter sp. (bae et al. 1996) . our results further indicate that hydroquinone is not the ring fission substrate because no enzymatic activities for hydroquinone dioxygenase or hydroxymuconic semialdehyde dehydrogenase were found. instead, upon conversion of hydroquinone by strain if1, we observed the formation of a transient metabolite that was identified as hydroxyquinol, indicating that a second hydroxylation takes place before ring fission. the production of hydroxyquinol could be due to the action of a separate hydroquinone monooxygenase, or it could be caused by a second hydroxylation step by the 4-fp monooxygenase. conversion of hydroquinone to hydroxyquinol before ring fission was also suggested for strains arthrobacter that degrade 4-nitrophenol (perry and zylstra 2007) and 4-chlorophenol (nordin et al. 2005) . ring fission of hydroxyquinol would produce maleylacetate, which we did not detect as an intermediate in 4-fp degradation, but a transient metabolite was found with mass properties identical to those of 3oxoadipate, which is expected to be the next intermediate in hydroxyquinol degradation. most cometabolic transformations of fluorophenols described in the literature involve the initial action of a phenol hydroxylase that results in the formation of fluorocatechols, which are subsequently transformed into fluoromuconates through a catechol 1,2-dioxygenase. the conversion usually proceeds with lactonization and elimination of fluoride (boersma et al. 2001; bondar et al. 1999; finkelstein et al. 2000; boersma et al. 1998; bondar et al. 1998 ). our results suggest that arthrobacter strain if1, which grows on 4-fp, possesses a new pathway for the degradation of 4-fp that involves immediate defluorination by a monooxygenase. strain if1 avoids the accumulation of possible toxic metabolites such as 3-or 4-fluorocatechol by this initial dehalogenation. biodegradation of 4-chlorophenol via a hydroquinone pathway by arthrobacter ureafaciens cpr706 19f nuclear magnetic resonance as a tool to investigate microbial degradation of fluorophenols to fluorocatechols and fluoromuconates 19f nmr metabolomics for the elucidation of microbial degradation pathways of fluorophenols 19 f nmr study on the biodegradation of fluorophenols by various rhodococcus species preferential oxidative dehalogenation upon conversion of 2-halophenols by rhodococcus opacus 1g selection of conserved blocks from multiple alignments for their use in phylogenetic analysis the ribosomal database project (rdp-ii): sequences and tools for high-throughput rrna analysis adaptation of alcaligenes eutrophus b9 and pseudomonas sp. b13 to 2-fluorobenzoate as growth substrate identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in rhodococcus opacus 1cp microbial breakdown of halogenated aromatic pesticides and related compounds bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt the metabolism of p-fluorobenzoic acid by a pseudomonas sp fluorine-containing natural products utilization of aromatic compounds by the penicillium strain bi 7/2 unspecific degradation of halogenated phenols by the soil fungus penicillium frequentans bi 7/2 degradation of 4-chlorophenol via the meta cleavage pathway by comamonas testosteroni jh5 how good is fluorine as a hydrogen bond acceptor? fluoride elimination from substrates in hydroxylation reactions catalyzed by p-hydroxybenzoate hydroxylase degradation of halogenated aliphatic compounds by xanthobacter autotrophicus gj10 a two-component monooxygenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from 4-nitrocatechol in bacillus sphaericus js905 fluorinated organics in the biosphere isolation of pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols arthrobacter nitroguajacolicus sp. nov., a novel 4-nitroguaiacol-degrading actinobacterium design and evaluation of useful bacterium-specific pcr primers that amplify genes coding for bacterial 16s rrna effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in arthrobacter chlorophenolicus a6 evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate identification of large linear plasmids in arthrobacter spp. encoding the degradation of quinaldine to anthranilate cloning of a gene cluster involved in the catabolism of p-nitrophenol by arthrobacter sp. strain js443 and characterization of the p-nitrophenol monooxygenase enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4-fluorobenzoate the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools metabolism of 4-chlorophenol by azotobacter sp. gp1: structure of the meta cleavage product of 4-chlorocatechol purification and properties of pentachlorophenol hydroxylase, a flavoprotein from flavobacterium sp. strain atcc 39723 a monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions confirmation of oxidative dehalogenation of pentachlorophenol by a flavobacterium pentachlorophenol hydroxylase acknowledgments this work was supported in part by the european community human potential programme under contract hprtn-ct-2002-00213 [biosap]. we thank erik de vries for 4fluorocatechol preparation and piet wietzes for technical assistance.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012439-28vi4c2j authors: abalakina, elena g.; tokmakova, irina l.; gorshkova, natalya v.; gak, evgueni r.; akhverdyan, valerii z.; mashko, sergey v.; yomantas, yurgis a. v. title: phage mu-driven two-plasmid system for integration of recombinant dna in the methylophilus methylotrophus genome date: 2008-11-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1696-7 sha: doc_id: 12439 cord_uid: 28vi4c2j a phage mu-driven two-plasmid system for dna integration in escherichia coli genome has been adjusted for methylophilus methylotrophus. constructed helper plasmids with broad-host-range replicons carry thermo-inducible genes for transposition factors mua and mub. integrative plasmids that are only replicated in e. coli could be mobilized to m. methylotrophus and contained mini-mu unit with a short terminus of mu dna, mu-attl/r. mini-mu unit was integrated in the m. methylotrophus genome via mobilization of the integrative plasmid to the cells carrying the helper in conditions of thermo-induced expression of mua and mub. in this system, mini-mu unit was mainly integrated due to replicative transposition, and the integrated copy could be amplified in the m. methylotrophus chromosome in the presence of helper plasmid. a kan-gene flanked by frt sites was inserted in one of the mini-mu units, and it could be readily excised by yeast flp recombinase that is encoded by the designed plasmid. the multiple mu-driven gene insertion was carried out by integration of the bacillus amyloliquefaciens α-amylase gene followed by curing the km(r) marker before integration of the second mini-mu unit with pseudomonas putida xyle gene encoding catechol 2,3-dioxygenase (c23o). methylotrophs are a diverse group of organisms that are capable of growing on single-carbon substrates, such as methane or methanol, as sole sources of organic carbon and energy. the availability of the raw material and its relatively low price make methylotrophic bacteria interesting candidates as producers of numerous biologically active compounds. therefore, these microorganisms are currently becoming more and more widely used in the manufacturing of natural and recombinant proteins (bélanger et al. 2004; fitzgerald and lidstrom 2003) , polysaccharides, amino acids, and vitamins (bourque et al. 1995; korotkova et al. 2002; motoyama et al. 1993 motoyama et al. , 2001 . the industrial application of methylotrophs has focused attention on the investigation of genetic regulation of c-1 metabolism (anthony 1993) . however, the majority of these bacteria have been poorly studied, and the genetic tools required for their investigation are limited. the methylophilus methylotrophus that is the subject of our investigation is especially hard to manipulate genetically because the majority of amino acids fail to penetrate in and export out of its cells, and therefore, the conventional methods for obtaining auxotrophic mutants are inapplicable under the circumstances. on the other hand, existing data on efficient expression of a few escherichia coli genes in m. methylotrophus (see for example gunji and yasueda 2006; tsujimoto et al. 2006 ) permit the hope of practical application of well-developed genetic systems to these methylotrophs. along with design of plasmid vectors, improvement of transposon mutagenesis is one of the priority genetic techniques for cloning and gene expression in methylotrophic bacteria (koch et al. 2001; lidstrom 2001, 2004) . for example, a possibility of using tn7 for sitespecific integration of recombinant dna in the genome of methylobacterium extorquens was demonstrated (choi et al. 2006) . the system can be applied to integration in a unique site in the recipient bacterial genome (koch et al. 2001) . in order to allow multiple integration of several copies of gene of interest and/or many dissimilar genes in different points of bacterial genome, the use of a wellinvestigated, both in vivo and in vitro, mobile genetic element, bacteriophage-transposon mu (mizuuchi 1983) , seemed most promising. it is known that mu employs two transposition mechanisms depending on the stage of the vital cycle: (1) simple insertion upon infection and introduction of its linear dna, practically, into the random point of the bacterial genome and (2) replicative transposition via formation of cointegrates at the lytic development and amplification of phage dna (craigie and mizuuchi 1985; sokolsky and baker 2003) . although some undetermined details that are being intensely investigated (see for example yin et al. 2007) remain in the mechanism of mu transposition, efficient systems for integration of recombinant dna in vitro (haapa et al. 1999 ) and in vivo in e. coli (akhverdyan et al. 2007; groisman and casadaban 1987) and salmonella typhimurium (lawes and maloy 1995) chromosomes have already been designed on its basis and are rather actively being used. in the present study, we have shown that it is also possible to perform the mu-driven integration of recombinant dna in the chromosome of m. methylotrophus. to this end, first of all, helper plasmids with thermoinducible genes encoding transposition factors of phage mu (mua and mub) were constructed on the basis of broad-host-range replicons of incp and incq incompatibility groups. integrative vectors, plasmids with constructed mini-mu units that are capable of mobilization transfer to m. methylotrophus cells, were also designed. mini-mu units from the integrative vectors flanked by mu-attl and mu-attr dna fragments were transposed in the chromosome of m. methylotrophus during the induced synthesis of mua and mub. mini-mu unit contained kanamycin resistance gene (kan) flanked with frt sites that permitted the selection of integrants for their resistance to kanamycin (km) and then to eliminate the marker from the bacterial chromosome exploiting the saccharomyces cerevisiae flp-frt-mediated site-specific recombination system (senecoff et al. 1985) adjusted to expression in m. methylotrophus. bacterial strains, plasmids, and cultivation conditions strains and plasmids used in the study are shown in table 1 . cells of m. methylotrophus as1 were grown at 37°c on a mineral medium seiia (gunji et al. 2004 ) of the following composition: k 2 hpo 4 , 1.9 g; nah 2 po 4 ×2h 2 o, 1.56 g; (nh 4 ) 2 so 4 , 5 g; mgso 4 ×7h 2 o, 200 mg; cacl 2 ×2h 2 o, 72 mg; cuso 4 ×5h 2 o, 5 μg; mnso 4 ×5h 2 o, 25 μg; znso 4 ×7h 2 o, 23 μg; fecl 3 ×6h 2 o, 9.7 mg/l, methanol 2%, ph 7.0. bactoagar (1.2%, difco, usa) and methanol 1% were applied to the solid media. m. methylotrophus as1 was resistant to chloramphenicol (cm), thus permitting the employment of this antibiotic to the counterselection of donor e. coli s17-1-based strains in bacterial mating experiments (see below). antibiotics to maintain plasmid dna in m. methylotrophus were added at the following concentrations: ampicillin (ap), 100 μg/ml; tetracycline (tc), 2 μg/ml; and streptomycin (sm), 50 μg/ml. km (10 μg/ml) and cm (20 μg/ml) were used for selection of the integrants and counter-selection of the donor, respectively. selection of clones containing the amplified mini-mu {[frt-km r -frt]-sm r } unit in the m. methylotrophus chromosome was performed on agar medium seiia with the sm content of 2 mg/ml. the e. coli strains were cultured at 37°c on liquid or solid luria-berthani (lb) medium; 1.2% of bactoagar was added in the latter case (sambrook and russell 2001) . the antibiotics (ap, 200 μg/ml; tc, 20 μg/ml; and sm, 50 μg/ml) were added during growth of the appropriate e. coli plasmid strains. all manipulations with strains m. methylotrophus as1 and e. coli with plasmids containing thermo-inducible genes, mua, mub, or flp, were carried out at 30°c unless induction was not required. selection of the in vitro constructed recombinant plasmids was conducted in the strains e. coli tg1 and e. coli s17-1. the treatment of recombinant dna and the southern hybridization was carried out in accordance to conventional protocols (sambrook and russell 2001) . preparations of restrictases, t4 dna ligases, and dna polymerase i klenow fragment from fermentas (lithuania) were used. taq dna polymerase (fermentas) or accutaqla dna polymerase (sigma) were used in accordance with the manufacturer's instructions to provide polymerase chain reaction (pcr) for confirmation the chromosomal modifications. the primers p1:5′-ttagatttggtggggcttgc→3′; p2: 5′-gtttatcagcttgctttcgagg→3′ were used. the biotin deca-label™ kit and biotin chromogenic detection kits (fermentas) were used to label and detect dna probes in southern hybridization. construction of recombinant plasmids paet7 (ap r , sm r ) was obtained on the basis of payc32 (chistorerdov and tsygankov 1986 ) by cloning of bamhi fragment from puc-muab (akhverdyan et al. 2007) . ptp310 (tc r ) was a result of cloning the same bamhi fragment of puc-muab in broad-host-range replicon prk310, a derivative of plasmid rp4/rk2 of incpα group (ditta et al. 1985; pansegrau et al. 1994) . pmiv5 (tokmakova et al. 2007 ) containing a multiplecloning site (mcs) flanked with transcription terminators between mu-attl and mu-attr was used as a vector for construction of the integrative plasmids. in order to obtain pmiv5-mob, the blunted bamhi fragment (~1.9 kb) of psup5011 (simon et al. 1983 (simon et al. , 1984 containing orit, traj, and trak from plasmid rp4 (pansegrau et al. 1990 ) was cloned into the pvuii site of pmiv5 located outside of mini-mu unit in the region that is nonessential for replication ( fig. 1 ). after that, the blunted 1.5 kb hindiii-ndei fragment of pkd4 (datsenko and wanner 2000) containing kan flanked with frt sites was cloned into ecorv site of pmiv5-mob to produce pmiv5-[frt-km r -frt]-mob (fig. 2) . three integrative plasmids were constructed using pmiv5-[frt-km r -frt]-mob as a vector: -the blunted 2.9-kb bglii-bamhi fragment of prt5 (smirnova et al. 1988 ) containing the amye gene for αamylase of b. amyloliquiefaciens was inserted into the smai site of the vector to obtain pmiv5-[frt-km r -frt]-amy-mob; -the ecori dna fragment of px1918 (schweizer 1993) containing the structural part of the xyle gene of the tol plasmid of p. putida mt-2 (inouye et al. 1981 ) was inserted into the ecori site of the vector to put the cloned gene under the transcriptional control of km r and create pmiv5-[frt-km r -frt]-xyl-mob; -the 1.9-kb ecorv fragment of php17 (tokmakova, unpublished) containing strab under control of a weak promoter p17 of m. methylotrophus was inserted into a smai site of the vector dna to make pmiv5-[frt-km r -frt]-sm r -mob (fig. 2) . the flp recombinase expressing plasmid pflp31 was constructed on the basis of paycter3 (gulevich et al. 2006) derived from payc32 (chistorerdov and tsygankov 1986) by cloning the bamhi-smai fragment (3.3 kb) of pcp20 dna (datsenko and wanner 2000) containing the gene flp of s. cerevisiae that was transcribed from promoter λp r governed by the plasmid encoded λcits857. the pflp31 can be eliminated from more than 30% of cells of m. methylotrophus after growth for 20 generations at 30°c without antibiotic on seii medium containing 25-30 μg/ml of acridine orange. integrative and helper plasmids were transferred into strain m. methylotrophus as1 by biparental mating using e. coli s17-1 bearing the respective plasmid as the donor. the overnight donor and recipient cultures (5×10 8 -10 9 cell/ml) were mixed at the ratio 1:10, harvested by centrifugation, washed with 0.15 m nacl, and placed on plates with the seiia medium containing 10% of lb broth and 0.5% of methanol. after 3-4 h of incubation at 30°c, the conjugation mixture was washed off from the plates with 1 ml of 0.15 m nacl and seeded in a dilution on agar seiia medium that contained the appropriate antibiotic (ap, tc, or sm) for selection of m. methylotrophus plasmid-carrier variants and cm for donor e. coli strain counter-selection. integration of mini-mu unit in the chromosome transposition of mini-mu unit into the m. methylotrophus chromosome was performed by conjugative transfer of integrative plasmid to the recipient strain m. methylotrophus that contained a helper plasmid. conjugation was carried out at 37°c to induce the synthesis of the transposition factors. the km r integrants were selected elimination of [frt-km r -frt] marker from the chromosome pflp31 was transferred into strain m. methylotrophus containing the integrated mini-mu unit with [frt-km r -frt] marker by mobilization (using plasmid-encoded ap r as the selective marker), and the km r ap r colonies were isolated. the isolated colonies were suspended up to the final concentration of 10 7 cell/ml in 5 ml of liquid medium seiia with ap, and the suspension was heated at 42°c for 20 min with the following incubation on a shaker at 37°c for 16-18 h to induce the flp recombinase synthesis. the culture was then plated on the non-selective seiia medium to obtain individual colonies. the latter were analyzed for the occurrence of the km and ap markers on the solid seiia medium with the appropriate antibiotic. detection of activities of α-amylase and c23o in recombinant m. methylotrophus strains a modified method of insoluble starch (amylopectin azure) hydrolysis was used for the qualitative analysis of αamylase activity (mantsala and zalkin 1979) . the replicas of the m. methylotrophus::{[frt-km r -frt]-amy} integrants were applied to petri dishes containing the solid seiia medium (1.5% amylopectin azure) and incubated at 37°c. visual detection of starch hydrolysis zones that correlated with α-amylase-active colonies was performed on the third and fourth day. the activity of catechol-2,3-dioxygenase (c23o) in the m. methylotrophus::{[frt-km r -frt]-xyle} cells was determined by the appearance of bright yellow color after colonies were sprayed with a solution of catechol (100 mm; zukowski et al. 1983 ). amplification of mini-mu unit in the chromosome of m. methylotrophus ptp310 was conjugatively transferred into strain m. methylotrophus::{[frt-km r -frt]-sm r } using tc r as a marker. after overnight incubation at 37°c on a shaker cell cultures were plated on the agar seiia medium containing different amounts of sm, and clones resistant to the high concentration of sm (2.0 mg/ml) were selected. the helper plasmid was eliminated from the selected clones during aerobic cultivation in the liquid seiia medium at 30°c for 24 h. the presence of additional copies of mini-mu unit in the chromosomal dna of m. methylotrophus resistant to high concentration of sm was confirmed by southern dna hybridization. the helper plasmid puc-muab was constructed and used earlier as a component of the two-plasmid system for mu transposition in e. coli (akhverdyan et al. 2007) . a gene for the temperature-sensitive repressor, cts62, and the genes for transposition factors mua and mub were located in this plasmid. however, being constructed on the puc-like replicon, puc-muab could only be replicated and maintained in e. coli cells. therefore, a modification of the helper plasmid was necessary in order to use the system in m. methylotrophus. two plasmids were constructed on the basis of broadhost-range replicons of different groups of incompatibility and were capable of being transferred to the methylotrophs by mobilization. one of them, paet7 (ap r , sm r , incq group; guerry et al. 1974) , was stably maintained in m. methylotrophus (percentage of cells that lost the plasmid within 20 generations under non-selective conditions at 30°c was no greater than 2%). paet7 could be eliminated with the frequency of no less than 30% by growing the m. methylotrophus in a medium with acridine orange (25-30 μg/ml) for 24-48 h. the second helper plasmid, ptp310 (tc r , incpα-group; ditta et al. 1985; pansegrau et al. 1994) , was maintained in the m. methylotrophus only under strictly selective conditions and qualitatively lost from the population when aerobically cultured (30°c, 48 h) in liquid medium without tc. the second component of two-plasmid system for mudriven transposition is integrative vector. to increase the efficiency of vector transfer into variety of microorganisms, the fragment containing the rp4-specific mob site was cloned into pmiv5 containing mu-attl/r sites. then, km r flanked by the frt sites was introduced in mini-mu unit as a marker to give pmiv5-[frt-km r -frt]-mob (see "materials and methods"; figs. 1 and 2) . the obtained paet7 and ptp310 can be used as helper plasmids, and pmiv5-[frt-km r -frt]-mob, in turn, as a mobilizable integrative plasmid with a selective marker, in the testing of mu-driven integration in m. methylotrophus cells. mu-driven transposition of km r in the m. methylotrophus chromosome and flp-mediated curing the marker the helper and integrative plasmids were applied to transformation of e. coli s17-1 (tra + ), and the obtained strains were used in succession as donors in the conjugation crossings with m. methylotrophus as1. this methylotrophic strain was resistant to cm, thus permitting the employment of this antibiotic to the donor counterselection. at the first stage, the helper plasmid paet7 was introduced in m. methylotrophus (with the efficiency 10 −2 -10 −3 relative to the initial number of the donor strain cells) by mobilization using sm r marker. at the second stage, m. methylotrophus as1 that contains a helper plasmid was used as recipient in crossing with e. coli s17-1/pmiv5-[frt-km r -frt]-mob. mobilization was performed at 37°c, ensuring the thermo-induced expression of mua and mub. the km r integrants were selected with the frequency of 10 −2 -10 −3 that correlated with the efficiency of plasmid mobilization, i.e., transposition of km r marker occurs practically in each cell of the recipient strain that received the integrative plasmid. later on, after paet7 was cured in the km r cells, the occurrence of mini-mu unit in the bacterial chromosome was confirmed by genetic analysis (ap s ) and pcr (using the mentioned primers p1 and p2 in "materials and methods," we detected the dna fragment of about 2,200 bp that corresponded to the size between mu-attl/r sites in the used km r -mini-mu unit). since both integrative and helper plasmids had the same ap r marker, the ap r km r cointegrates that could be formed in the process of replicative transposition would be genetically undistinguishable from the km r integrants obtained due to the simple insertion and containing the ap r helper plasmid (see fig. 3 ). during the long procedure required for the paet7 elimination, the cointegrates could have resolved, giving the same km r phenotype, as the products of simple insertion lost the helper plasmid. therefore, using paet7 as the helper plasmid, it was impossible to estimate which of the known mechanisms was involved in the mini-mu unit transposition into the genome of m. methylotrophus. an attempt to detect the cointegrates was carried out using another helper, ptp310, that did not contain ap r and carried tc r as a selective marker. after mobilization of pmiv5-[frt-km r -frt]-mob into m. methylotrophus as1/ptp310 and selection of km r methylotrophic clones, they were examined for the ap resistance. only 30% of these clones had phenotype km r , ap s , i.e., they were resulted from either the simple insertion or replicative transposition event, having the cointegrates already resolved. the remaining 70% of the analyzed clones had the km r , ap r phenotype being true cointegrates (fig. 3) . further cultivation of these clones in km-containing medium (without ap) led to the selection of km r ap s variants that were the products of cointegrate resolution. in this work, kan gene flanked with the frt regions was used as the selective km r marker for mini-mu unit integration. to excise this marker out of the m. methylotrophus chromosome, the pflp31 plasmid bearing the flp recombinase of s. cerevisiae under the control of the thermo-inducible promoter λp r was constructed. the the cointegrate can subsequently be resolved by a reciprocal recombination event between two mini-mu units fig. 4 visual detection of activities of α-amylase (starch hydrolysis zones) and c23o (bright yellow color) in the colonies of m. methylotrophus integrants pflp31 was transferred to the strain m. methylotrophus that contained mini-mu unit with km r marker in the chromosome, and flp synthesis was thermo-induced. after culture plating, 99% of the selected ap r clones had the km s phenotype. the km r marker excision was confirmed using pcr (using primers p1 and p2 in pcr we detected the decrease of the amplimers' size from about 2,200 to 815 bp after the marker excision). pflp31 was eliminated from the obtained km s clones. the resulting markerless strain can be used as recipient for further modifications. firstly, the gene for α-amylase (amye) of b. amyloliquefaciens was cloned into pmiv5-[frt-km r -frt]-mob, and the resulting mini-mu unit was integrated into the m. methylotrophus chromosome using km r as the selective marker. after the flp-frt-mediated km r excision was performed, expression of amye gene was detected in the obtained strain by zones of hydrolysis on a starchcontaining medium (fig. 4) . secondly, a structural part of the xyle gene in the tol plasmid of p. putida mt-2 was cloned into pmiv5-[frt-km r -frt]-mob under transcriptional control of the kan promoter. the mini-mu unit obtained was integrated into m. methylotrophus as1 and m. methylotrophus as1:: amy+. the expression of amye and xyle in the double integrant strain was demonstrated (fig. 4) . integration of mini-mu unit in the m. methylotrophus chromosome occurs mainly by replicative transposition; therefore, this mechanism can be employed for the amplification of the integrated genes. mini-mu unit {[frt-km r -frt]-sm r } was used for amplification as a model. it was already known that a single copy of strab under the control of p17 promoter can provide the resistance of m. methylotrophus to 500 μg/ml of sm, whereas methylotrophic cells that contained this gene on a moderate-copy-number plasmid were resistant to >2 mg/ml of sm (tokmakova, unpublished) . in the presence of ptp310 in conditions of induced mua and mub synthesis, the clones containing amplified mini-mu {[frt-km r -frt]-sm r } units were selected on the plates containing 2 mg/ml of sm, but with rather low frequency-2×10 −4 . the fact that the elevated resistance of the selected clones was due to the mini-mu unit amplification was proven by the southern hybridization (fig. 5 ). the selected clones had at least two copies of mini-mu unit in their genomes. unfortunately, the presence of two mini-mu units in the chromosome has already provided resistance to such high antibiotic concentrations (up to 10 mg/ml) that selection of variants with higher copy number was made impossible. 1 however, the possibility of mini-mu unit intrachromosomal amplification in the presence of mu transposition factors was demonstrated. it seems probable that this amplification based on the known intrachromosomal replicative transposition resulted in cointegrate formation leading to chromosomal rearrangements due to the inversion of the chromosomal fragment located between two obtained copies of transposon (metzler 2001) . the combined action of replication and site-specific recombination between two copies of transposon is necessary to restore the original position of the first copy during the transposition which 1 the real reason of nonlinear dependence of the level of cell resistance to sm on the copy number of mini-mu unit in m. methylotrophus chromosome is unclear. one of the possibilities could be a limitation of antibiotic transport facility, and so at the determined sm r genes expression level, the corresponding protein products could degrade all sm penetrated in the cell independently on extracellular sm concentration. genomic dna, prepared from 15 colonies selected on 2.0 mg/ml of sm and from the control strains was digested with ecorv, separated by gel electrophoresis in agarose (right) and hybridized with the internal fragment of kan used as the probe (left). five of 15 samples are shown here. lane 1 dna ladder, lane 2 a control clone with non amplified mini-mu unit, lanes 3-7 clones with amplified mini-mu unit, lane8 a negative control a wild-type clone seems less probable in the absence of the specific counterselection against chromosomal rearrangements. thus, in our experiments, we were unable to detect clones with the restored positions of the original copy of mini-mu unit in the chromosome: both chromosomal dna fragments carried mini-mu unit and selected by southern dna hybridization in new strains differed in size from the corresponding fragment in the strain progenitor (fig. 5) . in case of restored position of the original mini-mu unit, the new fragment had to be added to the previously detected one at the hybridization's pattern of the new strains. therefore, all the clones tested in the present study underwent chromosomal rearrangements. in the present study, the earlier constructed two-plasmid system for mu-driven integration of recombinant dna fragments in the e. coli chromosome (akhverdyan et al. 2007 ) was adjusted for efficient exploitation in m. methylotrophus. helper plasmids that are capable of autonomous replication in the methylotrophic cells and carry thermoinducible genes for the mu transposition factors are the first elements of the modified system. introduction of these plasmids into m. methylotrophus can be performed by electroporation (data not shown) and conjugative transfer. the use of helpers based on replicons of different stability broadens the experimental potential of the system for genetic modification of strains. for example, the helper paet7 which is stably maintained is suitable to use for multiple consecutive integrations of various mini-mu units in the same strain or for the several cycles of amplification of an integrated copy of mini-mu unit by the replicative transposition mechanism. at the same time, the use of the unstable helper, ptp310, is useful for one-cycle integration with rapid selection of a plasmidless recombinant strain. the second element of the system is the integrative vectors that can be replicated in only e. coli strains. these can be introduced into m. methylotrophus by conjugative transfer or by electroporation and possess mcs in mini-mu unit for cloning the genes of interest. the kan gene flanked with the frt sites provides the selection of the mini-mu unit integrants and can be excised if necessary from the final strain using pflp31 plasmid bearing the flp recombinase gene of s. cerevisiae. the advantage of the system for consecutive integration of a few heterologous genes in the m. methylotrophus chromosome was demonstrated using the amye gene from b. amyloliquefaciens and xyle gene from p. putida mt-2 as models. the amplification of the integrated genes in the m. methylotrophus genome using the mechanism of replicative transposition was also demonstrated, although the observed efficiency of amplification was significantly lower than that under analogous conditions in e. coli (akhverdyan et al. 2007 ). the following modification of the constructed integrative plasmid due to insertion of the small (about 400 bp) dna fragment carrying phage mu enhancer sequence (yin et al. 2007 ) in the mini-mu unit significantly increased efficiency of the possible intrachromosomal amplification: in the same conditions as described in the corresponding part of "results", we could detect the strains with two to six copies amplified mini-mu units (tokmakova et al., in preparation) . an adaptation of the mu transposition system for use in the m. methylotrophus was based on the previously known functional homology of dna-bending proteins from various bacteria and e. coli proteins hu and ihf (swinger and rice 2004) that are involved in formation of the transpososome structure along with phage-specific dna elements (mu-attl/r) and transposition factors (mua and mub; gueguen et al. 2005) . a priori, the simple insertion rather than the replicative transposition mechanism would operate most likely in m. methylotrophus because the former occurs via reparative instead of replicative dna synthesis, and therefore, lower amounts of host-specific proteins are necessary. however, the predominant formation of cointegrate structures observed in the experiments on primary mini-mu unit transposition and intrachromosomal mini-mu unit amplification support the latter statement. in other words, it was experimentally shown that the m. methylotrophus proteins, (1) can function in the same way as their analogs in e. coli cells during transpososome formation and (2) ensure all the following stages of the mini-mu unit replicative transposition. this makes it possible to employ mu-driven integration and amplification of integrated recombinant dna to practical applications. doubtless, the study of basic aspects of mu-driven transposition in m. methylotrophus requires further experiments. however, the designed system is already being exploited in the modification of m. methylotrophus strains, and experiments on metabolic engineering (tokmakova et al. 2007 ) focused on new biologically active and biotechnologically important substance production. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. development of the mini-mu system providing an effective integration and amplification of the genetic material in the escherichia coli chromosome methanol dehydrogenase in gram-negative bacteria production of heterologous protein by methylobacterium extorquens in high cell density fermentation high cell density production of poly-beta-hydrobutyrate (phb) from methanol by methylbacterium 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topologically unique interwrapped transpososome chromogenic identification of genetic regulatory signals in bacillus subtilis based on expression of a cloned pseudomonas gene key: cord-012090-pnt4y7zd authors: marasabessy, ahmad; moeis, maelita r.; sanders, johan p. m.; weusthuis, ruud a. title: enhancing jatropha oil extraction yield from the kernels assisted by a xylan-degrading bacterium to preserve protein structure date: 2011-05-10 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3312-5 sha: doc_id: 12090 cord_uid: pnt4y7zd we investigated the use of bacterial cells isolated from paddy crab for the extraction of oil from jatropha seed kernels in aqueous media while simultaneously preserving the protein structures of this protein-rich endosperm. a bacterial strain—which was marked as mb4 and identified by means of 16s rdna sequencing and physiological characterization as either bacillus pumilus or bacillus altitudinis—enhanced the extraction yield of jatropha oil. the incubation of an mb4 starter culture with preheated kernel slurry in aqueous media with the initial ph of 5.5 at 37 °c for 6 h liberated 73% w/w of the jatropha oil. since mb4 produces xylanases, it is suggested that strain mb4 facilitates oil liberation via degradation of hemicelluloses which form the oil-containing cell wall structure of the kernel. after mb4 assisted oil extraction, sds-page analysis showed that the majority of jatropha proteins were preserved in the solid phase of the extraction residues. the advantages offered by this process are: protein in the residue can be further processed for other applications, no purified enzyme preparation is needed, and the resulting oil can be used for biodiesel production. jatropha curcas is a well-known plant for the high fat and protein content of its seed ranging between 45% and 55% w/w and 20-30% w/w of the kernels, respectively (gubitz et al. 1999; lestari et al. 2010 ). this oil is economically attractive due to its potential application in biodiesel (lin et al. 2003; martinez-herrera et al. 2006 ). in addition, the kernel contains 28% protein, which has been extensively studied for food and non-food application (gubitz et al. 1999; lestari et al. 2010; lin et al. 2003; martinez-herrera et al. 2006 ). lestari extracted more than 80% of the protein from the kernels and addressed some potential applications of the isolated protein in various fields such as adhesives, coatings, and chemicals (lestari et al. 2010) . therefore, with respect to the overall economy of jatropha cultivation, it is interesting to find a commercial use for both oil and protein. protein recovery from the kernel requires aqueous extraction; hence, it is interesting to see if aqueous extraction can also be used for the release of oil. the common method of oil production from oilseeds as feedstock for biodiesel involves pressing of seeds and oil purification (degumming, deacidification, dewaxing, dephosphorization, dehydration, etc.) . these processes, together with esterification/transesterification, contribute to over 70% of the total biodiesel production costs (shuit et al. 2010; zeng et al. 2009 ). aqueous oil extraction (aoe) uses water as medium to facilitate oil liberation from oilseeds. aoe eliminates organic solvent consumption and so improves process economy (barrios et al. 1990; rosenthal et al. 1996) . aoe also enables several purification steps such as degumming, deacidification, dewaxing, and dephosphorization to be carried out simultaneously within the extraction step (caragay 1983 ) resulting in a more efficient process. we demonstrated earlier that thermophilic strains isolated from the gut of paddy crabs, one of which was identified as bacillus licheniformis, enhanced oil liberation up to 60% from aqueous j. curcas kernel, most likely via protein degradation (marasabessy et al. 2010) , which would be disadvantageous for protein recovery. in the present report, we confirmed that these thermophilic bacteria degraded extracted j. curcas protein. we also examined if preheating the kernels degraded the proteins in comparison to non-heated kernels by using sds-page analysis. next, isolation and selection of mesophilic bacteria from the gut of paddy crabs were performed based on their ability to liberate oil from j. curcas preheated kernel slurry. the aim was to obtain other microorganisms able to liberate oil without affecting the protein structures. the best strain was used for aqueous oil extraction from j. curcas kernel. the molecular weight distribution of protein in the residue (water phase and solid phase) after microbial treatment was also investigated to examine protein integrity. the quality of recovered oil was analyzed and compared with those of standard values of feedstock for biodiesel. jatropha seeds were harvested from j. curcas planted in serpong, indonesia (geocoordinates 6°21′31″ s, 106°40′ 33″ e). kernels were obtained after removal of the shells. the sun-dried kernels were stored at 4°c until used. paddy crabs were collected from bunds of a paddy field located in pamulang, indonesia (geocoordinates 6°20′52″ s, 106°42′ 20″ e). all chemical reagents, unless otherwise specified, were of analytical grade. the kernels (500 g) were autoclaved at 121°c for 15 min and then dried at 60°c overnight. the kernels were milled and sieved through a strainer with 1.0 mm pore diameter. to prepare the preheated kernel slurry, 25 g milled kernel was homogenized with 125 g purified water (milli-q) for 5 min using a waring blender. the weight ratio of solid material to water in the slurry was 1:5. under constant stirring-to keep the slurry homogenous-12 g of kernel slurry (equivalent to 2 g kernel) was used for the extraction of oil. protein extraction was carried out by extracting 1 g of sample with 30 ml solvent for 30 min in 50 ml capped centrifuge tubes. the mixing was conducted at room temperature by using a rotary mixer. the extracting solvents were water, nacl 1.0 m, and naoh 0.055 m as described previously (lestari et al. 2010) . solid-liquid separation was conducted at 4,000×g for 15 min by using a sorvall6+ centrifuge. a mixture of 15 g/l agar-agar (merck) and 10 g/l of j. curcas seed protein having a purity of ca. 83% w/w or 10.0 g/l of casein (merck) in water was boiled to dissolve agar. after autoclaving (121°c, 15 min), 15 ml proteinagar solution was aseptically poured in a sterile petri dish and brought to solidify overnight. the wells in protein-agar media were made by using a sterile rubber cork having a diameter of 9 mm. two milliliters of a 24-h old bacterial starter culture was centrifuged at 20,000 rpm for 5 min. the supernatant was filtered through a 0.22-μm bacterial filter (millipore), after which 50 μl of the filtrate (bacterial crude extract) was pipetted into the well. the plates were placed at 4°c overnight to let the extract absorb into the protein-agar media, followed by incubation at 37°c for 6 h and at 45°c for 6 h. clear zones surrounding the well indicating protein solubilization (degradation) by bacterial proteases were observed. a thermostable bacterial neutral protease from bacillus thermoproteolyticus (protex 14 l, genencor) at 200x dilution was used as the positive control, while the preheated bacterial crude extracts and preheated protex 14 l (100°c, 10 min), respectively, were used as negative controls. the crab paste was prepared as described previously (marasabessy et al. 2010) . to extract oil, 2.0 g of crab paste was mixed with 30.0 g of kernel slurry and incubated in a orbital shaker at 37°c, 150 rpm for 24 h. antibiotics were applied in some samples as described previously and the extracted oil was assayed gravimetrically (marasabessy et al. 2010 ). for the isolation of bacteria, one loop of crab paste was streaked out aseptically on a nutrient agar (na) medium plate (merck) and incubated at 37°c for 24 h. well separated colonies were picked up, subcultured, and maintained on na slants (37°c for 24 h). under constant stirring, 12 g of preheated kernel slurry (equivalent to 2 g kernel) was weighed out in a 100-ml flask. this was inoculated with 2 ml bacterial suspension, prepared by suspending cells of a bacterial culture grown on an na agar slant (in a tube having 1.5 cm diameter and 12 cm length) with 2 ml sterile water. the mixture was incubated at 37°c and 150 rpm for 24 h using an innova 44 incubator shaker (new brunswick), after which it was centrifuged at 7,400×g for 15 min. the extracted oil was assayed gravimetrically (marasabessy et al. 2010) . control experiments were performed using exactly the same treatment, however without bacterial inoculation. a bacterial strain showing the best performance was identified by partial sequence of 16s rdna as well as physiological tests conducted by dsmz (germany). bacterial starter culture was prepared as described previously (marasabessy et al. 2010) , except that the nutrient broth medium (nb, merck) was initially supplemented with 1.0% w/v milled jatropha kernel before autoclaving. to extract the oil, 12.0 g of jatropha kernel slurry was inoculated with 1.0 ml of the bacterial starter culture. antibiotics were applied in some samples as described previously (marasabessy et al. 2010) . the mixture was shaken at 150 rpm and 37°c. after incubation, the slurry was centrifuged at 7,400×g for 15 min. the free oil on the surface of the liquid in the centrifuge tube was assayed gravimetrically as reported previously (marasabessy et al. 2010 ). for xylanase detection, 15 μl bacterial crude extract was pipetted into a well (5 mm diameter) in an agar plate containing 0.2% remazol brilliant blue xylan (rbb-xylan, sigma) (strauss et al. 2001) . for cellulose detection, 50 μl bacterial crude extract was pipetted into a well (9 mm diameter) in an agar plate containing 0.4% carboxymethyl cellulose (cmc). the plates were placed at 4°c overnight to let the extract absorb into the agar media, followed by incubation at 37°c for 6 h (rbb-xylan agar) and 48 h (cmc agar). the cmc agar plate was stained with 0.03% congo red, followed by destaining with 1 m hcl (teather and wood 1982) . the clear zones surrounding the well indicate the hydrolysis of xylan and cellulose. molecular weight distribution of proteins was analyzed by using sds-page (nupage electrophoresis system with nupage novex bis-tris gels 10% from invitrogen). assay of total oil content, oil yield, and oil quality the total oil content of the oilseeds was determined by soxhlet method (aoac 1984) . the total oil content was 0.47 kg/kg jatropha kernels. this amount was taken as 100% recovery of oil in the calculations of jatropha oil yield in the extraction experiments. the free fatty acid and moisture content of the extracted oil was assayed by the titration method and the karl fischer method, respectively (aoac 2002) . the oxidative stability index (osi) was assayed using 873 biodiesel rancimat apparatus from metrohm. the effect of thermophilic crab bacteria on jatropha protein integrity in our previous publication, some thermophilic bacteria (namely bk21, bk22, and b. licheniformis strain bk23 isolated from paddy crabs) extracted up to 60% of the jatropha oil from non-heated kernels after 24 h incubation at 50°c under non-optimized conditions (marasabessy et al. 2010) . from the protein-agar plate experiment (fig. 1) , we found that those strains hydrolyzed both j. curcas kernel protein and casein upon incubation at 50°c. bk21 showed the highest protease activity and b. licheniformis bk23 the lowest, as indicated by the size of the clearing zone diameter, for both types of protein. the bright clear zones proved that proteins available in the kernel were completely solubilized. the effect of heat pretreatment on jatropha protein integrity because we wanted to study the effect of crab's gut bacteria working at lower temperature on the extraction yield of oil from j. curcas kernels, internal factors within the kernels influencing oil liberation had to be minimized. since the kernels contain microorganisms as well as seed enzymes which might interfere with the crab bacteria involved in oil liberation, we applied two different heat pretreatments on kernels, at 105°c or 121°c for 30 min, to deactivate enzymes and to kill microorganisms before being used for oil extraction. the proteins were extracted from the kernels and the extracts were subjected to sds-page analysis (fig. 2) . the solubility of proteins in water depends on various factors such as ionic strength and ph; therefore, 1 m nacl and 0.055 m naoh were also used as extractants besides water (lestari et al. 2010) . the protein pattern of the heat-treated kernels extracted with nacl and naoh was identical with that of the untreated kernels, showing that heat treatment at both 105 and 121°c for 30 min did not affect the protein composition. compared to the other samples, the untreated sample extracted with water is missing three small bands at molecular weights of approximately 20, 23, and 25 kda, indicating that heat treatment increased the water solubility of the proteins. concluding, heat pretreatment did not have an effect on position and relative intensity of the different protein bands on the sds-page, indicating that no significant alteration of the chemical structures of the proteins occurred. we decided therefore to employ preheated kernels (by autoclaving at 121°c for 15 min) for oil extraction in the subsequent experiments. effect of paddy crab paste on oil extraction the presence of mesophilic bacteria in paddy crabs having a positive effect on jatropha oil liberation was determined by incubating 2 g paddy crab paste with 30 g preheated kernel slurry (containing 5 g kernel) at 37°c and 150 rpm for 24 h with and without addition of antibiotics (fig. 3) . oil extraction in control experiments to which no crab paste was added resulted in 7% oil after 24 h. addition of antibiotics to control samples also gave a low oil yield (4%). incubation of preheated kernel slurry with crab paste and antibiotics significantly improved the oil yield to 62%. it is evident that paddy crab paste exhibits a strong effect towards oil liberation from jatropha kernel. furthermore, it was shown that excluding antibiotics from the crab-kernel sample resulted in even higher oil liberation (70%). the significant yield improvement from 62% to 70% indicates that mesophilic bacteria derived from paddy crabs take part in the entire mechanism of jatropha oil liberation from preheated kernel. based on these results, we decided to isolate mesophilic bacteria living in the gut of paddy crabs as our experimental strains for microbial jatropha oil extraction. protease, xylanase, and glucanase activity of bacterial crude extract we isolated 20 colonies of mesophilic crab bacteria, but we selected only seven colonies for further testing, namely strains mb4, mb5, mb7, mb11, mb12, mb13, and mb20 based on differences in colony form and microscopic observation. the detection of protease activity in crude extract revealed that mb4 is the only strain exhibiting protease, with different strengths of activity against the two types of protein tested: casein and j. curcas protein (fig. 4a, b) . the mb4 protease showed strong activity against casein as shown by a bright clear zone in the casein layer due to casein degradation by hydrolysis (fig. 4a) . however, the mb4 protease did not function with j. curcas protein under the conditions tested, as shown by the absence of a clear zone formed in the j. curcas protein layer (fig. 4b) . xylanase activity was found only in mb4 crude extract as shown by formation of a clear zone in rbb-xylan agar medium (fig. 4c) . congo red staining in cmc agar medium showed a negative result for glucanase activity in the crude extract of all strains tested (fig. 4d) . figure 4d depicts that the enzyme gc-220 (genencor inc, usa) lacked glucanase activity as no clear zone formed in cmc agar medium. selection of paddy crab bacterial strain for jatropha oil extraction figure 5 shows the amount of oil extracted from 12 g preheated jatropha kernel slurry (containing 2 g kernel), inoculated with the isolated bacterial strains directly prepared by suspending the na culture slant with 2 ml water. we found that mb4 gave the highest jatropha oil yield (63%), a 15-fold increase compared to a control experiment containing antibiotics. mb4 was selected for further tests at different conditions of incubations. the phenotypical characterization conducted by dsmz (germany) indicates that mb4 is a bacillus pumilus strain ( table 1 ). the partial sequencing of 16s rdna (data not shown) conducted also by dsmz shows a similarity of 100% to bacillus altitudinis and 99% to the type strain of b. pumilus. the partial sequence has been submitted to the genbank (accession number hq860795). considering the result of the partial sequencing, a clear identification to species level is not possible. further examinations will be required to find out the novelty of the strain mb4. the the optimization of microbially assisted oil extraction was conducted in three steps. in first instance, we incubated preheated jatropha kernel slurry with the starter culture of strain mb4 at 37°c over 24 h in order to find the optimum incubation time (fig. 6a) . second, we studied the effect of initial ph (4.5, 5.5, 6.5, 7.5, and 8.5) of the kernel slurry on oil liberation by strain mb4; kernel slurry ph was adjusted to the desired value by using 4 m sodium hydroxide or 4 m sulphuric acid solutions before inoculation of bacteria starter cultures (fig. 6b) . third, we optimized incubation temperature (37, 45, 50, and 55°c) for jatropha oil extraction (fig. 6c) . figure 6a shows that the oil yield in the control samples (containing antibiotics) remained below 10% throughout incubation for 24 h. the addition of mb4 starter culture to preheated kernel slurry resulted in a sharply increased oil yield to about 60% (tenfold increase compared to the control experiment) only within 6-8 h, after which it remained constant until 24 h. based on these results, we decided to shorten the incubation time to 6 h in the subsequent experiments of microbial-assisted oil extraction. the oil yield of kernel slurry incubated with mb4 for 6 h at different ph values (4.5, 5.5, 6.5, 7.5, and 8.5 ) is shown in fig. 6b . the oil yield of mb4-treated sample increased from 65% at ph 4.5, to peak at 73% at ph 5.5, and then decreased to 50% at ph 8.5. contrary to the curve trends obtained with mb4, the oil yield of control sample (containing antibiotics) decreased rapidly from 40% at ph 4.5 to 10% at ph 5.5, and then increased to 20% at ph 8.5. as a conclusion, strain mb4 has an optimum initial ph of 5.5 at 37°c. based on these results, we therefore studied the effect of incubation temperature on oil liberation by mb4 at ph 5.5 for 6 h. the oil yield from kernel slurry incubated with strain mb4 for 6 h at ph 5.5 and different temperatures is shown fig. 6c . it is evident that the highest extraction yield of 73% was obtained at an incubation temperature of 37°c. the oil yield of mb4-treated sample decreased from 73% to 60% as the temperature increased from 37°c to 45°c. the oil yield of mb4-treated sample slightly increased to 64% when the temperature increased from 45°c to 55°c. the oil yield of the control sample showed a slow increasing trend from 10% (37°c), reaching a maximum oil yield of 30% only at 55°c. this slow increase can at least partially explain the increasing trend of oil liberation in the mb4-treated sample at temperature in the 45-55°c range. we investigated the molecular weight distribution of protein in liquid phase (supernatant) and solid phase (cake) after mb4 oil extraction, in comparison to those extracted with 0.055 m naoh, by using sds-page analysis as shown in fig. 7 . we did not recover protein in the interfacial phase for sds-page analysis because we observed a very low amount of solid in the interfacial phase (between oil-water) after centrifugation, indicating a lower amount of oil-water emulsion after mb4 treatment. figure 7 shows that almost all proteins in the range of 1.0 to 88.5 kda available in 0.055 m naoh-extracted sample were also available in the solid phase, with the exception of one protein (88.5 kda) that was missing in the solid phase. three additonal proteins of 14.7, 27.4, and 44.9 kda that were not available in naoh-extracted sample were found in the solid phase as well as in the liquid phase. six proteins of 1. 7, 8.5, 9.4, 10.6, 11.3, and 32 .1 kda that fig. 7 sds-page analysis of mb4-treated jatropha kernel proteins. bands of proteins from duplicated samples of water phase (1 and 2), solid phase/cake (3 and 4) in comparison to jatropha protein (5 and 6). s stands for standard of protein marker fig. 6 incubation of preheated jatropha kernel slurry with mb4 (white circle) and without mb4 (white square): (a) oil extraction yield after incubation (37°c for 24 h), the initial ph was not adjusted; (b) oil extraction yield after incubation at different initial ph (37°c for 6 h); and (c) oil extraction yield after incubation at ph 5.5 at different temperatures (37, 45, 50, and 55°c for 6 h) were available in naoh-extracted sample were not detected in the liquid phase. furthermore, 13 additional proteins of 2. 8, 11.9, 13.5, 14.7, 20.1, 25.1, 27.4, 29.2, 41.3, 44.9, 59.3, 100.1, and 130 .4 that were not available in naoh-extracted sample were found in the liquid phase. the quality of oil after aqueous oil extraction oil quality data in table 2 show that the oil obtained by mb4 extraction, in general, meet the german fuel standard din v 51605 for pure plant oil (rapeseed oil), except for the acid value (av) which was found 8.6, or more than eight times higher than the standard value. j. curcas seed kernels have a high fat and protein content ranging between 45% and 55% w/w and 20-30% w/w, respectively (gubitz et al. 1999; lestari et al. 2010 ). the oil is investigated for its suitability as a biofuel, whereas the protein has been extensively studied for food and non-food application (gubitz et al. 1999; lestari et al. 2010; lin et al. 2003; martinez-herrera et al. 2006) . therefore, with respect to the overall economy of jatropha cultivation, it is important to find commercial outlets for both oil and protein. in studying the effect of heat pretreatment on protein integrity, we found proteins resolution on electrophoresis gel gave identical band positions among non-heated, preheated at 105°c for 30 min, and preheated at 121°c for 30 min (fig. 2) . this means that the structure of jatropha protein exhibits high thermal stability against thermal processing upon heating up to 121°c for 30 min. thermal properties of proteins are important to study the changes during heat processing which, in turn, are useful in the processing designs for protein-based products (horax et al. 2011) . aqueous extraction is necessary for the recovery of the protein from the kernel, and in order to decrease process costs it is therefore interesting to liberate the oil from the seed in the same step. in protease-assisted aqueous oil extraction from oilseeds, oil-bound proteins are hydrolyzed into smaller fractions, thereby altering their structure and functionality (moure et al. 2006) . similar studies in jatropha oil extraction reported previously did not highlight the importance of preserving protein structure during oil extraction process. if the protein structures are to be conserved to a large extent in the recovery of oil from oilseeds, the use of bacterial strains or enzymes liberating oil by other means than protein solubilisation is a reasonable choice. apart from proteases, a number of microbial enzymes have been studied to enhance oil extraction yields from oilseeds: amylase, glucanase, pectinase, cellulolytic, and hemicellulolytic enzymes (dominguez et al. 1994) . we were therefore interested to isolate and select other microbial strains from the crab's gut capable of assisting oil liberation without degrading protein. the paste of paddy fields crabs are traditionally used for coconut oil extraction in java. in a previous article, we have also applied paste crab to release oil from jatropha kernels (marasabessy et al. 2010 ). whereas we now were able to release 70% of the oil, we previously only liberated 30% of the oil. even though the experimental conditions in using paddy crab paste as the research material between the present study and the previous study (marasabessy et al. 2010 ) look similar, they are not entirely the same for two reasons. first, in our present study, preheated kernels were used as substrate instead of non-heated kernel used in the previous study. preheating the kernels might have enhanced the dissolution of cell components which were previously bound to the original structures of cells (williams 2005) , allowing crab's enzymes or microbial enzymes to have access in breaking oil barriers, resulting in the release of more oil as compared to control experiments (fig. 3) . second, the different batch of crab paste used in the present study might have resulted in differences in oil liberation. we found that mb4 starter culture was able to extract 73% oil from jatropha kernel slurry when incubated for 6 h at 37°c and ph 5.5. this is in good agreement with the jatropha oil yield of 85.6% and 74% extracted by using protease of alcalase (novo nordisk, denmark) and protizyme (jaysons agritech, india), respectively (shah et al. 2005; winkler et al. 1997) . the use of viscozyme (novo nordisk, denmark) as a hemicellulase/cellulase formula gave a comparable oil yield of 70% (winkler et al. 1997) . we have shown that protease from strain mb4 bears no activity against jatropha protein. hence, by considering the optimal ph and temperature of mb4 (ph 5.5 and 37°c, respectively) and also the presence of xylanase in the crude extract of mb4, it is most likely that the strain mb4 facilitates oil liberation at 37°c via the degradation of hemicellulose that forms the oil-containing cell wall structure of the kernel (rosenthal et al. 1996) . bacterial identification results suggested the strain mb4 as b. pumilus or the closely related b. altitudinis. in case of b. pumilus, previous investigations have reported the potential application of b. pumilus as xylanase producer (ahlawat et al. 2007; battan et al. 2007; kapoor and kuhad 2007; kapoor et al. 2008; nagar et al. 2010; wang et al. 2010; yasinok et al. 2010) . in contrast, we found that only a few publications are available on the potential application of b. altitudinis. after mb4-assisted oil extraction, the extracted oil has an av below 14% (table 2) , which seems applicable for biodiesel production since a chemical pretreatment to reduce the acid value from 14% to 1% before transesterification of jatropha oil into biodiesel has been established recently, which results 99% yield of biodiesel (tiwari et al. 2007) . concluding, strain mb4 identified as b. pumilus or b. altitudinis isolated from paddy crab liberated 73% w/w of jatropha oil from preheated kernel in aqueous system after 6 h incubation at 37°c. it is suggested that the strain mb4 facilitates oil liberation via degradation of hemicellulose. incubation of j. curcas kernel with strain mb4 preserves the jatropha protein structure to a large extent. mb4assisted oil extraction has several advantages: (a) no purified cocktail enzyme preparation is required, (b) protein integrity is mostly preserved, and (c) this method results in jatropha oil with a quality which is suitable for biodiesel production. production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp official methods of analysis optimization of an enzymatic process for coconut oil extraction enhanced production of cellulase-free thermostable xylanase by 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toxic/antimetabolic constituents, and effects of different treatments on their levels, in four provenances of jatropha curcas l. from mexico functionality of oilseed protein products: a review production and optimization of cellulase-free, alkali-stable xylanase by bacillus pumilus sv-85s in submerged fermentation aqueous and enzymatic processes for edible oil extraction extraction of oil from jatropha curcas l. seed kernels by combination of ultrasonication and aqueous enzymatic oil extraction reactive extraction and in situ esterification of jatropha curcas l. seeds for the production of biodiesel screening for the production of extracellular hydrolytic enzymes by non-saccharomyces wine yeasts use of congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen biodiesel production from jatropha (jatropha curcas) with high free fatty acids: an optimized process an alkali-tolerant xylanase produced by the newly isolated alkaliphilic bacillus pumilus from paper mill effluent recovery of oils and fats from oilseeds and fatty materials enzyme-supported oil extraction from jatropha curcas seeds xylanase from a soil isolate, bacillus pumilus: gene isolation, enzyme production, purification, characterization and one-step separation by aqueoustwo-phase system rapid in situ transesterification of sunflower oil acknowledgments the authors gratefully acknowledge the koninklijke nederlandse akademie van wetenschappen, scientific programme indonesia-netherlands (spin-knaw) for the financial assistance. we also would like to acknowledge erna subroto, phd student at rijks universiteit groningen the netherlands for conducting oil quality analysis, and dianika lestari for her help in conducting gel electrophoresis of jatropha protein of our samples.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-011320-cwvkox29 authors: puente-massaguer, eduard; lecina, martí; gòdia, francesc title: integrating nanoparticle quantification and statistical design of experiments for efficient hiv-1 virus-like particle production in high five cells date: 2020-01-06 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-10319-x sha: doc_id: 11320 cord_uid: cwvkox29 the nature of enveloped virus-like particles (vlps) has triggered high interest in their application to different research fields, including vaccine development. the baculovirus expression vector system (bevs) has been used as an efficient platform for obtaining large amounts of these complex nanoparticles. to date, most of the studies dealing with vlp production by recombinant baculovirus infection utilize indirect detection or quantification techniques that hinder the appropriate characterization of the process and product. here, we propose the application of cutting-edge quantification methodologies in combination with advanced statistical designs to exploit the full potential of the high five/bevs as a platform to produce hiv-1 gag vlps. the synergies between cci, moi, and toh were studied using a response surface methodology approach on four different response functions: baculovirus infection, vlp production, vlp assembly, and vlp productivity. toh and moi proved to be the major influencing factors in contrast with previous reported data. interestingly, a remarkable competition between gag vlp production and non-assembled gag was detected. also, the use of nanoparticle tracking analysis and flow virometry revealed the existence of remarkable quantities of extracellular vesicles. the different responses of the study were combined to determine two global optimum conditions, one aiming to maximize the vlp titer (quantity) and the second aiming to find a compromise between vlp yield and the ratio of assembled vlps (quality). this study provides a valuable approach to optimize vlp production and demonstrates that the high five/bevs can support mass production of gag vlps and potentially other complex nanoparticles. electronic supplementary material: the online version of this article (10.1007/s00253-019-10319-x) contains supplementary material, which is available to authorized users. virus-like particles (vlps) have emerged as promising nanoparticles, particularly in the field of new recombinant vaccines (mohsen et al. 2017) . compared with other conventional vaccines such as live-attenuated or inactivated viruses, vlps can elicit potent antibody and t cell responses in a safe manner since they do not contain genetic material from the virus itself (andersson et al. 2018 ). among the different proteins able to self-assemble as vlps, the group-specific antigen (gag) polyprotein has demonstrated a wide applicability in different research areas. these include their use as immunogens (young and ross 2006) , drug delivery vehicles (deo et al. 2015) , scaffolds for surface antigen presentation (vidigal et al. 2018) , and research surrogates . a common need in all these applications is the understanding of the production system itself, which is paramount to define the most adequate conditions for vlp production. the baculovirus species autographa californica multiple nucleopolyhedrovirus (acmnpv) has been successfully used for the production of a variety of vlp types. the ease of handling, the high productivities associated and the capacity to accommodate large quantities of dna have made the baculovirus expression vector system (bevs) a very attractive platform (fernandes et al. 2013; gómez-sebastián et al. 2014) . from the different available insect cell lines, high five electronic supplementary material the online version of this article (https://doi.org/10.1007/s00253-019-10319-x) contains supplementary material, which is available to authorized users. cells have been reported to produce high vlp titers with a reduced level of contaminant baculovirus (senger et al. 2009 ). in fact, cervarix®, the first vlp-based human papillomavirus vaccine approved, is produced using the high five/bevs. recent advances in the field of nanoparticle quantification and characterization have brought the opportunity to deepen into the understanding of the bevs. in the frame of initiatives such as process analytical technologies (pat), the application of new and more sophisticated characterization technologies will be a powerful tool to increase the understanding of how the process conditions influence the quality of the final product. nanoparticle tracking analysis (nta) and flow virometry are gaining interest as tools to monitor the production of different particles, including vlps (nika et al. 2019; pereira aguilar et al. 2019) . to date, most of the studies dealing with vlp production are performed with indirect quantification methods based on monomer detection, which hinder the appropriate assessment of the final product and the yields obtained (haynes et al. 2009; pillay et al. 2009; visciano et al. 2011 ). in addition, the simultaneous production of extracellular vesicles (ev), which frequently fall in the size range of vlps, is not often considered and may lead to an erroneous estimation of vlp titers. with the aim to deepen in the characterization of the different existing nanoparticle populations, the egfp was fused in frame to the gag gene from the human immunodeficiency virus serotype 1 (hiv-1). considering the information from previous reports, we decided to apply these novel techniques for optimizing the gag vlp production process using the high five/bevs platform. different methods are available for process improvement, from the classical one-variable-at-a-time approach to more sophisticated statistical design of experiments (doe) (puente-massaguer et al. 2019) . so far, vlp production studies have focused either on the former approach (krammer et al. 2010; pushko et al. 2017) or on simple factorial designs (pillay et al. 2009; pastor et al. 2019) , limiting the analysis of higher order effects between the variables influencing vlp production. this is of special relevance since the use of lower or higher moi than strictly required might reduce the maximum vlp yield obtained or be detrimental to the system, respectively. here, we applied a response surface methodology (rsm) to evaluate the impact of cell concentration at infection (cci), multiplicity of infection (moi), and time of harvest (toh) on vlp production. to go a step further in process understanding, the inclusion of several responses during process optimization has shown successful results and applicability in different research areas (pinzi et al. 2011; honary et al. 2014; bukzem et al. 2016) . particularly, the three responses considered in addition to vlp production were vlp assembly, baculovirus infection, and vlp productivity. these are of special relevance in processes with recombinant baculovirus since it is desirable to define production conditions encompassing high production yields with an acceptable ratio of correctly assembled gag in the form of vlps, but at the same time maintaining high productivities. a multiple-criteria decision analysis (mcda) was implemented to combine the rsm-optimized responses into a global optimal set of conditions based on two criteria: quantity and quality. the two optimal production conditions were successfully validated, and the quantity optimum was compared with other gag vlp production strategies using the high five/bevs. finally, the correct formation and morphology of the vlps produced was characterized using cryogenic transmission electron microscopy (cryo-tem). the results presented in this work represent an advance with respect to current literature related to gag vlp production in the high five/bevs and provide useful insights into nanoparticle-based process characterization. high five (cat. num. b85502, thermo fisher scientific, grand island, ny, usa) and sf9 cells (cat. num. 71104, merck, darmstadt, germany) were cultured in suspension in the lowhydrolysate content and animal origin-free sf900iii medium (thermo fisher scientific). both cell lines were routinely maintained at exponential growth phase in 125-ml disposable polycarbonate erlenmeyer flasks (corning, steuben, ny, usa) in 15 ml of medium and subcultured three times a week at a density of 2-4 × 10 5 cells/ml for high five cells (puente-massaguer et al. 2018 ) and 4-6 × 10 5 cells/ml for sf9 cells (puente-massaguer et al. 2020) . all cultures were shaken at 130 rpm using an orbital shaker (stuart, stone, uk) and maintained at 27°c in an incubator. cell count and viability were performed using the nucleocounter nc-3000 (chemometec, allerod, denmark) according to manufacturer's instructions. the recombinant autographa californica multicapsid nucleopolyhedrovirus (acmnpv), containing the hiv-1 gag matrix protein fused in frame to egfp (bv-gagegfp) (hermida-matsumoto and resh 2000) and under the control of the polyhedrin promoter, was constructed using the baculogold system (bd biosciences, san jose, ca, usa). baculovirus amplification was performed in the sf9 cell line (cat. num. 71104, merck, darmstadt, germany) cultured in sf900iii medium by infection at 3 × 10 6 cells/ml, a multiplicity of infection (moi) of 0.1, and harvest at 96 hours post infection (hpi). titration of baculovirus infectious particles was conducted using the plaque assay method. briefly, 2 ml of sf9 cells at a concentration of 0.5 × 10 6 cell/ml was plated per well in 6well plates (nunc, roskilde, denmark) and left to attach for 30 min at rt. then, the sf900iii medium was aspired and sf9 cells were infected with the addition of 1 ml per well of serial dilutions (10 −4 to 10 −9 ) of the bv-gagegfp samples. six well plates were incubated at very mild mixing conditions during 1 h to homogeneously distribute the baculoviruses. afterwards, baculovirus-containing supernatants were aspired and 2 ml of a type vii-a agarose (merck) solution warmed at 42°c was added per well to cover the infected cells. the agarose solution was prepared by mixing an ultrapure water solution containing 3% w/v agarose with sf900iii medium supplemented with 10% fetal bovine serum (fbs) at a 1:1 ratio. after agarose cooling, 2 ml of sf900iii medium supplemented with 10% fbs was added per well and the plates were incubated for 5 days at 27°c without agitation. the concentration of infectious baculovirus particles, expressed as plaque forming units (pfu), was measured by visual inspection after 3 h staining with a 0.3% w/v neutral red solution (merck) prepared in dpbs. cell growth and infection studies were performed in high five cell cultures seeded at 3 × 10 5 cell/ml in 15 ml and maintained at mid-exponential phase by cell passaging every 48 h. several moi, cell concentrations at infection (cci), and times of harvest (toh) were evaluated and the different parameter combinations were defined according to a doe-based approach. samples were taken every 12-24 h to monitor viable cell concentration, cell viability, baculovirus infection, intra-and extracellular gagegfp expression, and nanoparticle production. samples were harvested by centrifugation at 3000×g for 5 min and supernatants were maintained at 4°c until analysis. cell pellets were stored at − 20°c. the percentage of gagegfp expressing cells was assessed using a bd facs canto ii flow cytometer equipped with a 488-nm laser configuration (bd biosciences). briefly, 0.3 ml of infected cell cultures was harvested by centrifugation at 300×g for 5 min and fixed using 4% p-formaldehyde for 10 min. cells were then centrifuged at 500×g for 5 min to remove p-formaldehyde, resuspended in 0.3-ml ice-cold dpbs (thermo fisher scientific), and maintained at 4°c until further analysis. bv-gagegfp-infected high five cells were observed using a tcs sp5 confocal microscope (leica, wetzlar, germany). cells were stained with 0.05% v/v of cellmask™ and 0.1% v/v of hoechst (thermo fisher scientific) to visualize the lipid membrane and cell nucleus, respectively. a washing step prior to observation was performed by centrifugation at 300×g during 5 min, and then cells were resuspended in fresh sf900iii medium. samples were placed in 35-mm glass bottom petri dishes with a 14-mm microwell (mattek corporation, ashland, ma, usa) for visualization. the vlp production process was monitored during 6.5 min at 48 hpi using the resonant scanner mode of the confocal microscope (leica). the amount of gagegfp produced by high five infected cells was measured intra-and extracellularly by spectrofluorometry. supernatants of gagegfp producing cells were harvested by centrifugation at 3000×g for 5 min and maintained at 4°c until analysis. intracellular gagegfp content was evaluated by disruption of pelleted cells by means of three freeze-thaw cycles (2.5 h frozen at − 20°c and thawed at 37°c for 0.5 h). samples were vortexed for 5 s between cycles. lysed pellets were resuspended in 0.5 ml of tms buffer (50-mm tris-hcl, 150-mm nacl, 2-mm mgcl 2 , ph 8.0), centrifuged at 13,700×g for 20 min and kept at 4°c. gagegfp fluorescence was measured with a cary eclipse fluorescence spectrophotometer (agilent technologies, santa clara, ca, usa) at rt as follows: λ ex = 488 nm (5-nm slit), λ em = 500-530 nm (10-nm slit). the sf900iii medium and a 0.1 mg/ml quinine sulfate solution were used as control patterns to normalize rfu values in samples between different experiments. a nanosight ns300 (malvern panalytical, malvern, uk) was used to measure fluorescent and non-fluorescent particles by nanoparticle tracking analysis (nta). samples were diluted in 0.22-μm-filtered dpbs and continuously injected into the device chamber through a pump at an average concentration of 10 8 particles/ml (20-60 particles/frame). videos of 60 s from independent triplicate measurements were analyzed with the nta 3.2 software at rt. the nanoparticle number was evaluated in the 100-250-nm range. fluorescent and non-fluorescent nanoparticles were also analyzed using a cytoflex lx (beckman coulter, brea, ca, usa) equipped with a 488-nm blue laser for fluorescent particle detection and a 405 nm laser/violet side scatter configuration for improved nanoparticle resolution. samples were diluted in 0.22-μmfiltered dpbs and triplicate measurements from independent samples were analyzed with the cytexpert 2.3 software at rt. supernatants of bv-gagegfp-infected high five cells harvested at the different conditions of the doe were ultracentrifuged in a double cushion of 5 ml of 25% and 8 ml of 45% (w/v) sucrose (merck) solution prepared in dpbs and dmem (thermo fisher scientific), respectively. five milliliters of supernatants from triplicate experiments were loaded in ultracentrifuge tubes (beckman coulter) and filled to the top with sterile dpbs. sample ultracentrifugation was performed in a beckman optima l100xp centrifuge (beckman coulter) equipped with a sw-32ti rotor at 31,000 rpm for 2.5 h at 4°c. samples were taken from each ultracentrifuge fraction, and pellets were resuspended in 100 μl of dpbs and kept overnight at 4°c. all samples were stored at − 20°c until their measurement by spectrofluorometry and at 4°c for nanoparticle analysis by flow virometry. the same procedure was applied to characterize the optimal conditions obtained with the doe and the mcda approach. cryogenic transmission electron microscopy vlp morphology was assessed using a transmission electron microscope equipped for sample cryogenics (cryo-tem). briefly, 4 μl of sample was blotted onto emr holey carbon films on 400-mesh copper grids (micro to nano, wateringweg, netherlands) previously subjected to a glow discharge treatment in a pelco easiglow™ discharge cleaning system (pelco, fresno, ca, usa). deposited samples were then plunged into liquid ethane at − 180°c using a leica em gp workstation (leica) and observed in a jem-2011 tem operating at 200 kv (jeol ltd., akishima, tokyo, japan). a response surface methodology (rsm) was applied to assess the effect of the main factors influencing the bevs: cci (cell/ml), moi (pfu/cell), and toh (h) on different responses. the selected doe responses were baculovirus infection (% of infected cells), vlp production (fluorescent particle/ml), gagegfp polyprotein assembled in the form of vlps (% of gagegfp assembled as vlps), and vlp productivity (fluorescent particle/ml h). vlp production and vlp productivity responses were transformed to log values using the logarithmic regression to reduce the funnel shape effect of the residuals. this transformation compensated the increasing differences in standard deviation associated to higher figures, a common issue when simultaneously considering values with more than 1000-fold difference (barbur et al. 1994) . in parallel, baculovirus infection and vlp assembly responses were converted using the logistic regression to limit the possible values of the functions to the 0-100% range: where y n is the converted response and y n is the experimental response in natural values. a box-behnken design was used to determine the influence of cci, moi, and toh on each evaluated response. these variables were screened at a low (− 1), medium (0), and high level (+ 1) as indicated in table 1 . the different levels were linearly related for cci and toh and exponentially for moi, always in an equidistant manner. the results obtained for each response were fitted to a second-order polynomial by the least squares method (eq. 3): where y is the response variable, β 0 is the model intercept term, β 1 − β 9 are the model coefficients, and ε is the experimental error. fitting of the different equations based on eq. 3 was performed with the r software (r foundation for statistical computing, vienna, austria). three-dimensional plots were constructed to facilitate model interpretation. the most common doe approach involves the optimization of a single response. however, such analysis may not be enough for the bevs, since the definition of a production condition cannot be accurately predicted from an individual property. then, the objective of including more than one response in the study was to determine global conditions integrating different key aspects of the bevs. to this purpose, a multiple-criteria decision analysis (mcda) based on desirability functions was implemented. the different responses were transformed to a dimensionless desirability scale (d n ) ranging between 0 and 1: where y n is the predicted response of the fitted equation, d n is the desirability response function, w accounts for the weight value, and min n and max n are the minimum and maximum acceptable values of y n , respectively. the weight (w value) ns non-statistically significant depended on the relative importance assigned to each individual response. then, all the different desirability response functions under study were combined into a single equation, denoted as overall desirability (od): where od is the overall desirability function to be maximized and n is the number of responses studied in the optimization process. the experimental design and statistical analyses of the different equations were performed using r software. the quality of the regression of the fitted equations was evaluated with the r 2 and adjusted r 2 coefficients in percentage units. an analysis of variance (anova) f test was used to determine the significance of the equations and the individual coefficients were assessed with a t test. the lack-of-fit (lof) test was used to evaluate differences between experimental and pure error of the fitted equations. in all analyses, p values of 0.05 and 0.1 were considered statistically significant with 95% and 90% confidence, respectively. initially, the growth of high five cells was characterized to determine an adequate range of viable cell concentration at infection (cci) for the doe. cells maintained a high viability (> 90%) until 96 h of culture, coinciding with the peak of viable cell concentration. in these conditions, cells reached a maximum of 7.3 ± 0.2 × 10 6 cells/ml and a doubling time (t d ) of 18.7 ± 0.5 h (fig. 1) . considering the extent of the exponential phase and the t d , the range for cell concentration at infection was restricted to 0.5-2.5 × 10 6 cells/ml. some difficulties were encountered when defining the different levels of multiplicity of infection (moi) and time of harvest (toh) since very little information on the use of the bevs with high five cells in the sf900iii medium was available. the ranges for both variables were eventually fixed according to previous experience with this system (data not shown) and based on studies of the high five/bevs in other cell culture media (sander and harrysson 2007; pillay et al. 2009 ). the moi experimental range was fixed between 0.01 and 5 with the aim to screen low and high moi conditions in the same doe. in the case of toh, the boundaries were defined as 24-84 hpi. doe space boundaries did not involve high cci out of the exponential phase or medium exchange in order to simplify their industrial application, but these conditions were evaluated as a contrast to the optimal conditions obtained as further presented. combining rsm and mcda to define the optimal vlp production conditions a three-variable (cci, moi, and toh) box-behnken rsm was constructed and the following objective responses were analyzed: baculovirus infection efficiency, vlp production, vlp assembly, and vlp productivity. this experimental design was selected among different candidates due to its variance distribution and spatial properties ( (table 1) . the different datasets were adjusted to the second-order polynomial functions by the least squares method according to eq. 3. the statistical significance of the different functions and their associated coefficients was confirmed by anova (table 1) . each function was used to construct three-dimensional plots for a better comprehension of the synergies between cci, moi, and toh (fig. 2) . as expected, baculovirus infection progressed more rapidly with increasing moi (fig. 2a-c) . however, the pace of infection decreased when using the same moi at higher cci. a similar behavior was observed between vlp production and vlp productivity ( fig. 2d-i) . low moi required longer toh to achieve higher vlp yields, whereas higher moi shortened this period. in both cases, the best production and productivity titers were obtained in the 1.5-2.5 × 10 6 cell/ml cci range. toh also proved to be the most significant variable regarding the amount of gagegfp monomer assembled in the form of vlps (fig. 2j-l) . increasing toh and moi substantially decreased the ratio of vlps to non-assembled monomers, regardless the value of cci. the models were used to predict an optimal combination of cci, moi and toh to maximize each specific response. in this case, different independent variable combinations resulted in the optimum condition for each function (table 2 ), thus making difficult to define a global optimum condition combining all the different models at once. therefore, a multiplefig. 2 three-dimensional plots of the different functions based on the box-behnken experimental results. a-c baculovirus infection. d-f vlp production. g-i vlp productivity. j-l vlp assembly as a function of cci, moi, and toh. all the graphs were constructed by representing the effect of two independent variables on a response while maintaining the third one at a fixed level criteria decision analysis (mcda) based on desirability functions was applied in order to determine a global optimum combining the different functions. every model function was transformed to a dimensionless scale (eq. 4) and given a weight according to the importance of each response. a higher priority was assigned to vlp production, vlp assembly, and vlp productivity (w = 2), while a lower restriction was assigned to infection (w = 1). two possible response combinations were considered in the mcda, the first focused on maximizing product quantity and the second one maximizing product quality. to do this, the vlp assembly function was excluded from the mcda in the definition of a global optimal condition targeting product quantity. the application of the mcda for the product quality optimum resulted in a cci, moi, and toh of 1.1 × 10 6 cell/ml, 1 pfu/cell, and 33.6 hpi, respectively. the optimum maximizing product quantity was determined as 2.0 × 10 6 cell/ml for cci, a moi of 2.5 pfu/cell, and a toh of 69.3 hpi. the two global optimal conditions obtained by the combination of rsm and mcda were successfully corroborated in a validation experiment. high five cell growth was arrested after bv-gagegfp infection with > 80% of the culture infected at 24 hpi ( fig. 3a-b) . as expected, a higher cell viability was observed for the quality optima (33.6 hpi) compared with the quantity condition at toh (69.3 hpi). confocal microscopy analysis of infected cells showed successful colocalization (yellow) of the gagegfp polyprotein (green) with the cell membrane (red), thus indicating that vlp formation and production was taking place (fig. 3c) . this process could be tracked in real time using a confocal fluorescence microscope with the high-speed acquisition mode (fig. s1) . measurement of fluorescence indicated that the highest pace of gagegfp production was achieved with higher moi. also, the highest fluorescence yield in the supernatant was achieved with the quantity optimum (2448.6 ± 23.8 rfu) at 69.3 hpi (fig. 4a) . the toh for the quality optimum condition coincided with the peak of intracellular gagegfp production, but not for the quantity optimum. in all cases, gagegfp production in the supernatant progressively increased with longer toh, although the intracellular gagegfp levels reached a maximum concentration that subsequently decreased (fig. 4b) . analysis of the vlp production process of the quantity optimum by flow virometry evidenced that there was a continuous increase in vlp concentration (fig. 4c) . comparison of vlp production in both optima by means of nanoparticle tracking analysis (nta) yielded a 10fold and 4.8-fold increase in production and productivity in the quantity over the quality optimum, respectively. in all cases, the experimental outcome was within the predicted limits (table 3) . similar ratios of vlps to total nanoparticles were observed, representing a 9.7 ± 3.1% for the quality and a 9.3 ± 1.1% for the quantity condition. in contrast, analysis of the optima by sucrose cushion ultracentrifugation showed a 4.9-fold increase of gagegfp monomer assembled in the form of vlps in the quality optimum (64.6 ± 5.1%). the sn and sn -25% fractions, corresponding to non-assembled gagegfp, showed significantly larger levels of fluorescence in the quantity optimum (fig. 4d) . however, vlp-containing fractions (25-45% and 45%) were more enriched in the quality condition. bv-gagegfp titration of both optima by plaque assay resulted in a 2.7-fold increase of infectious particles in the quantity (13.2 ± 5.1 × 10 7 pfu/ml) compared with quality condition (4.8 ± 1.5 × 10 7 pfu/ml). an acceptable correlation between indirect vlp quantification with spectrofluorometry and direct nanoparticle measurement with nta was only achieved for the quality optimum condition (eq. 1). on the contrary, gagegfp fluorescence levels of the quantity optimum measured by spectrofluorometry (2448.6 ± 23.8 rfu) and their conversion to vlp concentration according to eq. 1 resulted in a 9.6-fold higher value than the one obtained by direct vlp quantification using nta. however, multiplying this fluorescence value by the vlp assembly factor calculated by analytical ultracentrifugation (table 3 ) allowed to determine the gagegfp fluorescence exclusively attributed to vlps (323.2 ± 3.1 rfu) and discard that of unassembled gagegfp monomer. introducing the fluorescence value associated to vlps in eq. 1 resulted in a better prediction to nta quantification (4.9 ± 0.5 × 10 10 vlp/ml). supernatant characterization of the quality and quantity optima by cryogenic transmission electron microscopy (cryo-tem) indicated that vlps were correctly formed in both conditions (fig. 5) . interestingly, the median vlp size was higher in the quality (181.4 ± 4.4 nm) compared with the quantity optimum (164.7 ± 0.3 nm). the presence of evs and baculoviruses was also detected, in agreement with the existence of a non-fluorescent particle population observed with nta. in addition to the optimization work performed, the quantity optimum condition was compared with two conditions used in other works for the production of high vlp titers in order to corroborate the results of the study. the first approach consisted in infecting high five cells at a high cell concentration and moi. to this purpose, high five cells were infected at 6 × 10 6 cell/ml with a moi of 5. the second strategy consisted in performing a medium replacement before infection using the quantity optimum. this option was considered as a means to value if the added effort of performing a medium replacement would provide a substantial advantage in vlp concentration. both conditions were not examined in the doe in order to avoid the added difficulties required at process level, but were compared with the quantity optimum as a contrast since they have been used in the literature. baculovirus infection progressed more rapidly after medium exchange than in the original condition but the drop in cell viability was not significantly affected ( fig. 6a-b) . on the other hand, baculovirus infection advanced more slowly in the high cci condition, to the point that cell culture was not fully infected after 72 hpi. medium exchange before infection increased gagegfp production by 1.3-fold at 69.3 hpi, corresponding to 3168.6 ± 100.2 rfu in comparison with the 2528.0 ± 76.3 rfu achieved in the quantity condition ( fig. 6c-d) . the same improvement was observed in vlp quantification by nta, with 4.3 ± 0.2 × 10 10 vlp/ml for medium exchange while 3.4 ± 0.3 × 10 10 vlp/ml was measured for the quantity optimum condition. however, baculovirus infection at the high cci condition reached a significantly lower gagegfp production (66.6 ± 3.3 rfu) and also in terms of vlp production (2.3 ± 0.6 × 10 8 vlp/ml). the median size of the vlps produced in these conditions did not substantially vary from the quantity optimum (168.2 ± 3.3 nm), being 169.6 ± 3.3 nm for the medium exchange and 160.5 ± 3.7 nm for the high cci. similar levels of total nanoparticles were obtained in the three different conditions, thus representing a big difference in the ratio of vlp to total nanoparticles (table 4 ). remarkable differences were also observed as regards the amount of infectious baculovirus particles produced. medium replacement before infection increased the levels by 2.1-fold (47.0 ± 9.9 × 10 7 pfu/ml) compared with tested. infected cells were measured every 24 h until their optimal toh. c high five cells infected with the bv-gagegfp at 2.0 × 10 6 cell/ml and a moi of 2.5 and observed at 48 hpi. cell nucleus was stained with hoechst (blue) and cell membrane with cellmask™ (red). the mean and standard deviation of triplicate experiments are represented the 22.7 ± 2.5 × 10 7 pfu/ml achieved in the quantity optimum condition. however, a much lower baculovirus titer of 0.9 ± 0.3 × 10 7 pfu/ml was obtained in the high cci condition. the high five/bevs has demonstrated a high versatility to produce a wide variety of recombinant proteins. however, the capabilities of the high five cell line as a production platform of complex multimeric structures, such as enveloped vlps, have not been explored in detail. to date, the assessment of vlp production has been performed based on indirect quantification methods (pillay et al. 2009 ). moreover, most of the research has been conducted either in low performing or animal compound containing cell culture media (krammer et al. 2010; wilde et al. 2014) . although these works have brought advances in the production of nanoparticles using the high five/bevs, a deeper study would contribute to exploit the full potential of such platform. cci, moi, and toh were selected as the critical parameters affecting the production of hiv-1 gag vlps in the bevs. for a comprehensive analysis, four different responses were considered and simultaneously evaluated by means of rsm and mcda. toh and moi were shown to be particularly relevant to achieve a high vlp concentration but also in fig. 6 comparison of vlp production using the quantity optimum with medium exchange and baculovirus infection at a high cci. a-b viable cell concentration, cell viability, and bv-gagegfp infection process in the different conditions tested. c-d supernatant and intracellular gagegfp production. the average of triplicate experiments is represented. medium exchange was performed prior to bv-gagegfp infection and consisted in infecting high five cells at 2.0 × 10 6 cell/ml with a moi of 2.5 pfu/cell. as regards the high cci condition, high five cells were infected at 6 × 10 6 cell/ml with a moi of 5 maintaining an adequate ratio of gagegfp assembly in the form of vlps. pillay and co-workers used a factorial experiment to assess the effect of different moi comprised in the range of 0.1-5 in high five cells (pillay et al. 2009 ). they did not identify any significant difference in gag yield, but here we observed that moi plays a significant role in vlp production. interestingly, including a function for vlp assembly based on the measurement of the gagegfp fluorescence distribution in the different fractions of an ultracentrifugation proved to be highly relevant. these results in combination with direct nanoparticle quantification by nta and flow virometry permitted to obtain a more accurate perspective of the process. a pronounced decrease in vlp assembly was shown with increasing toh, limiting a favorable combination with the other three responses. to this purpose, two different global optimization strategies were examined by mcda, one considering the vlp assembly function (therefore with priority on product quality) and the other not (therefore with priority on product quantity). remarkable differences were encountered between the two optima, achieving a 10-fold higher vlp production in the quantity condition, whereas a 4.9-fold increase in the assembly of gagegfp as vlps was obtained in the quality optimum. then, it is evident that high five cells possess a high capacity to produce gagegfp, but there is a remarkable competition between the formation of vlps and the release of unassembled gagegfp monomer. the large amount of gagegfp produced, potentially overcoming the capacity of the cell line itself to process all the gagegfp in the form of vlps, combined to cell membrane disruption could be the reason explaining these results. this could be appreciated even in the quality optimum itself, in which a high cell viability of 92.4 ± 0.6% was measured at harvest (33.6 hpi) (fig. 3a) and a 64.6 ± 5.1% of the fluorescence in the supernatant was associated to vlps, meaning that around the 35% remaining was due to unassembled gagegfp monomer. these results emphasize the need to complement the methods most currently used for vlp identification or quantification based on monomer detection with direct nanoparticle assessment techniques, specially using the high five cell line (maranga et al. 2003; pillay et al. 2009; tagliamonte et al. 2010; shoja et al. 2013; monteiro et al. 2016; fuenmayor et al. 2019 ). the application of cryo-tem helped to gain insight into the identification of the morphological features of the vlps produced (fig. 5) . cryo-tem has recently been highlighted as powerful methodology for nanoparticle characterization as the different specimens can be observed in native conditions (martin et al. 2016; maldonado et al. 2016; gallagher et al. 2018) . gagegfp vlps presented the expected size of 100-200 nm and morphology consistent with immature hiv-1 vlps, comparable with vlps produced in sf9 (nika et al. 2017 ) and hek 293 cells (venereo-sanchez et al. 2016 ). the rod-shaped structure corresponding to the infective form of the baculovirus was also detected. the presence of evs was observed alongside vlps, which are known to be produced as way of communication in eukaryotic cells (ratajczak et al. 2006) . evs were more heterogeneous in size (50-500 nm) and shape compared with vlps and less electron-dense. these results coincide with the measurements performed by nta and flow virometry where a remarkable population of non-fluorescent nanoparticles was detected. future efforts are needed to understand their role in insect cell lines since little information is available. a maximum hiv-1 gagegfp vlp concentration of 3.9 ± 0.1 × 10 10 vlp/ml was achieved in the supernatant using the product quantity optimum. thus, these conditions would be selected in case of designing a vlp manufacturing process. however, if the presence of baculoviruses posed a risk for the final vlp application, the quality optimum provides a 2.7-fold reduction in infectious particles, which would facilitate the downstream process. difficulties were encountered in the comparison of the vlp titer obtained since most of the studies producing gag vlps with the insect cell/bevs used indirect quantification methods based on monomer detection (buonaguro et al. 2001; pillay et al. 2009; visciano et al. 2011) . nevertheless, when the comparison of nanoparticle production was possible, this optimum condition proved to be superior to gag vlp production in hek 293 cells without ) and with additive supplementation (cervera et al. 2015a ) by 14.4 and 1.7-fold, respectively. moreover, these conditions proved to be superior to influenza vlp production using the sf9 (thompson et al. 2015) and high five/bevs (carvalho et al. 2016 ) by 6.7 and 25-fold, respectively. the potential of the quantity optimum was also evaluated against other baculovirus infection strategies in this work. infection at a high cci using a high moi resulted in a much lower yield of vlp production probably owing to the scarcity of some nutrients in the medium (table 4 ). bv production was also significantly reduced in such conditions. these differences in production could be related to the cell density effect previously described in insect cells with the bevs huynh et al. 2013; huynh et al. 2014 ). on the other side, medium replacement before bv infection moderately increased vlp production but duplicated baculovirus production. this strategy has proven to be an efficient manner to increase both the yield of recombinant proteins (caron et al. 1990 ) and baculoviruses ) with the sf9/ bevs cells and also in mammalian cells (bollin et al. 2011; cervera et al. 2015b) . in this case, no substantial benefit was obtained, thus indicating that the added efforts required to perform medium replacement might not be advantageous. in conclusion, a detailed study of gag vlp production using the high five/bevs was conducted. the application of rsm and mcda allowed to go a step further and capture the synergies between the influential factors affecting the vlp production process. the combination of advanced statistical methods with a variety of nanoparticle quantification and characterization techniques allowed to obtain a more comprehensive picture of the process and to determine a highyielding and reproducible condition for vlp production. the approach here proposed is valuable for studies dealing with the production of other complex nanoparticles. future efforts in transferring this process into bioreactor scale should allow to define a robust platform for vlp production. virus-like-vaccines against hiv cell density effect in the baculovirus-insect cells system: a quantitative analysis of energetic metabolism design of experiment in cho and hek transient transfection condition optimization optimization of carboxymethyl chitosan synthesis using response surface methodology and desirability function high efficient production of pr55gag virus-like particles expressing multiple hiv-1 epitopes, including a gp120 protein derived from an ugandan hiv-1 isolate of subtype a chemokine receptor interactions with virus-like particles baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange high-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system bioorthogonal strategy for bioprocessing of specific-site-functionalized enveloped influenza-virus-like particles generation of hiv-1 gag vlps by transient transfection of hek 293 suspension cell cultures using an optimized animal-derived component free medium selection and optimization of transfection enhancer additives for increased virus-like particle production in hek293 suspension cell cultures extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement chimeric virus-like particles made using gag and m1 capsid proteins providing dual drug delivery and vaccination platform insect cells as a production platform of complex virus-like particles extended gene expression for gag vlp production achieved at bioreactor scale characterization of hemagglutinin antigens on influenza virus and within vaccines using electron microscopy significant productivity improvement of the baculovirus expression vector system by engineering a novel expression cassette development and validation of a quantitation assay for fluorescently tagged hiv-1 virus-like particles optimized production of hiv-1 virus-like particles by transient transfection in cap-t 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system distinct particle morphologies revealed through comparative parallel analyses of retrovirus-like particles major findings and recent advances in virus-like particle (vlp)-based vaccines targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system expression of full-length her2 protein in sf9 insect cells and its presentation on the surface of budded virus-like particles an her2-displaying virus-like particle vaccine protects from challenge with mammary carcinoma cells in a mouse model defining the multiplicity and time of infection for the production of zaire ebola virus-like particles in the insect cell-baculovirus expression system at-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification optimization of chimeric hiv-1 virus-like particle production in a baculovirus-insect cell expression system multiple response optimization of vegetable oils fatty acid composition to improve biodiesel physical properties nanoscale characterization coupled to multi-parametric optimization of hi5 cell transient gene expression a statistical approach to improve compound screening in cell culture media application of advanced quantification techniques in nanoparticle-based vaccine development with the sf9 cell baculovirus expression system virus-like particles displaying h5, h7, h9 hemagglutinins and n1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication using cell size kinetics to determine optimal harvest time for spodoptera frugiperda and trichoplusia ni bti-tn-5b1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein enhanced papillomavirus-like particle production in insect cells development of a stable insect cell line constitutively expressing rotavirus vp2 constitutive expression of hiv-vlps in stably transfected insect cell line for efficient delivery system critical assessment of influenza vlp production in sf9 and hek293 expression systems hemagglutinin and neuraminidase containing virus-like particles produced in hek-293 suspension culture: an effective influenza vaccine candidate rmce-based insect cell platform to produce membrane proteins captured on hiv-1 gag virus-like particles generation of hiv-1 virus-like particles expressing different hiv-1 glycoproteins high five and sf9-evaluation of host-virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression elicitation of immunity to hiv type 1 gag is determined by gag structure publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declare that they have no competing interests.ethical approval this article does not contain any studies with human participants performed by any of the authors. key: cord-339694-sp212tai authors: jiang, xinpeng; hou, xingyu; tang, lijie; jiang, yanping; ma, guangpeng; li, yijing title: a phase trial of the oral lactobacillus casei vaccine polarizes th2 cell immunity against transmissible gastroenteritis coronavirus infection date: 2016-03-28 journal: appl microbiol biotechnol doi: 10.1007/s00253-016-7424-9 sha: doc_id: 339694 cord_uid: sp212tai transmissible gastroenteritis coronavirus (tgev) is a member of the genus coronavirus, family coronaviridae, order nidovirales. tgev is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. an oral lactobacillus casei (l. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) were repeated 20 times and the d antigenic site of the tgev spike (s) protein was repeated 6 times. immunization with recombinant lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. the first immunization is more important than the last immunization in the series. the recombinant lactobacillus elicited specific systemic and mucosal immune responses. recombinant l. casei had a strong potentiating effect on the cellular immunity induced by the oral l. casei vaccine. however, during tgev infection, the systemic and local immune responses switched from th1 to th2-based immune responses. the systemic humoral immune response was stronger than the cellular immune response after tgev infection. we found that the recombinant lactobacillus stimulated il-17 expression in both the systemic and mucosal immune responses against tgev infection. furthermore, the lactobacillus vaccine stimulated an anti-tgev infection th17 pathway. the histopathological examination showed tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev with an intestinal tropism in piglets. the tgev immune protection was primarily dependent on mucosal immunity. lactic acid bacteria (lab) are a group of gram-positive bacteria that includes species of lactobacillus, leuconostoc, pediococcus and streptococcus. consumed for centuries, lab has enjoyed a long and safe association with humans and animals for healthy food. over the past decade, there has been increasing interest in the use of lab as mucosal delivery vehicles. the application of lab stems from research into effective strategies to deliver vaccine antigens, which may come into contact with the mucosal tissues for the first time, such as the intranasal, oral and genital mucosal surfaces (lavelle and o'hagan 2006; malik et al. 2007 ). mucosal delivery of therapeutics or vaccines for chronic diseases and infections of mucosal origin could increase their potency and specificity. because the mucosal immune system builds an effective iga barrier in no more than 3 days, contact with the mucosal tissues will neutralize the pathogenic microorganism outside of the host. there are abundant studies in the field of lab vaccines. one major advantage of the use of lab as a delivery vehicle for vaccines is that the bacteria can elicit both antigen-specific secretory immunoglobulin (ig) a and an effective systemic immune response. the specific igas have the same function as the neutralizing iggs. some candidate lab vaccines elicited antigen-specific iga responses in faeces, saliva or bronchoalveolar and intestinal lavage fluids (wells and mercenier 2008) . additionally, lactobacilli are probiotics that may confer health benefits to the host (bengmark and gil 2006; corthesy et al. 2007; isolauri et al. 2002; saarela et al. 2000) , and there is accumulating evidence that lactobacilli are effective at preventing intestinal disease in both humans and animals due to their ability to inhibit pathogen adhesion to the intestinal wall and prevent inflammatory processes (blum and schiffrin 2003; de vrese and marteau 2007; ouwehand 2007; sartor 2005; sheil et al. 2007 ). because the porcine digestive tract is similar to the digestive tract of human infants with respect to anatomical and histological features and digestive physiology (kararli 1995; oswald et al. 2003; tadros et al. 2003) , the pig has been used as an animal model to study gastrointestinal diseases of human infants (gunzer et al. 2002) and vaccination studies against these diseases. coronaviruses (covs) comprise a large family of enveloped, positive-stranded rna viruses that infect a broad range of animal hosts as well as humans. well-known representatives are porcine transmissible gastroenteritis virus, porcine respiratory cov, porcine epidemic diarrhoea virus, canine cov, feline cov, bovine cov, human covs, severe acute respiratory syndrome-associated cov, murine hepatitis virus (mhv), avian cov infectious bronchitis virus (ibv) and turkey cov (tcov). the most famous and critical coronavirus is the middle east respiratory syndrome virus found in africa and asia (siddell et al. 1983 ). this study investigates whether mucosal immunization is effective in stimulating a protective immune response against cov infection. transmissible gastroenteritis coronavirus (tgev), which is a member of the genus coronavirus, family coronaviridae, and order nidovirales, is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs (cavanagh 1997) . the viral rna consists of a single strand comprised of three major structural proteins: a phosphorylated nucleoprotein (n protein) and two glycoproteins (the membrane (m) and the spike (s) proteins) (schwegmann-wessels and herrler 2006). the s protein has four sites (a, b, c and d). both the a and d sites were demonstrated to induce tgev-neutralizing antibodies (di-qiu et al. 2011) ; however, the a site is highly glycosylated and thus is not suitable for expression in the lab prokaryotic expression vector. additionally, the different tgev sites induce different immune responses. following infection with virulent transmissible gastroenteritis coronavirus, isolated mesenteric lymph node cd4+ t cells mounted a specific proliferative response against infectious or inactivated purified virus upon secondary in vitro stimulation (anton et al. 1996) . the peptide n321 defines a functional t helper epitope that elicits t cells capable of collaborating with b cells specific for different tgev proteins (anton et al. 1995) . the most important finding is that oral immunization with a recombinant lactobacillus vaccine and infection with tgev elicit various immune responses, such as humoral immunity and cellular immunity. here, an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study the mucosal immune response (jiang et al. 2014) . in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) are repeated 20 times and the d antigenic site of the tgev spike (s) protein is repeated six times. the pig model was developed to study intestinal mucosal immune responses (ruan and zhang 2013) . probiotic feed supplementation may benefit the animal host directly by preventing infection and combating the causative agent of the intestinal disorder by balancing the disrupted equilibrium of the enteric flora and augmenting the host's immune responses. however, lab vaccine has not received national law or certification for human or animal use against coronaviruses. the first clinical trial to use recombinant lab demonstrated that the containment strategy for l. lactis expressing recombinant il-10 was effective against crohn's disease (braat et al. 2006) . our laboratory has researched the lab vaccine for more than one decade, and we have developed many lab vaccines in the field of piglet diarrhoea (di-qiu et al. 2011; liu et al. 2011; qiao et al. 2009; yigang and yijing 2008) . this study is the last step to obtain new drug certification in china. this lactobacillus vaccine has been demonstrated to increase the treg population in the mouse model (jiang et al. 2014) . tregs effectively depressed t and b cell proliferation, and some studies demonstrated that this regulation also depressed proliferation in inflammatory bowel disease. our study investigated whether the recombinant lactobacillus vaccine gradually increased the breg and treg populations during the immunization process at the first step of immunization (unpublished data). the intestinal immune system of the pig maintains its ability to mount an active immune response against pathogens and exhibits tolerance to at least food antigens and probably commensal flora through an extremely complex network of cellular and humoral immune interactions. consequently, it is important to elucidate the immunological inductive sites of the protective mucosal immune response following oral immunization in pigs. this vaccine could induce tgev antibody immune responses in both the humoral and mucosal immune systems. mdp and tuftsin possess substantial immunopotentiating properties and can induce cellular-mediated immune responses upon oral administration in mice. however, their use in oral vaccines against tgev challenge in the pig host may have different results. furthermore, the only host (and target of the vaccine) of the transmissible gastroenteritis coronavirus is the weaning piglet. there have been no clear reports concerning whether tgev infection stimulates th1 or th2type immune response. the relationship between humoral and cellular immune responses is not clear; moreover, whether the systemic or mucosal lymphoid response will be the primary immune response following oral immunization is unknown. at present, we are not certain which pathway the lactobacillus vaccine will provoke against tgev infection. our study is the first to analyse immunity in response to oral immunization and tgev infection. virus, bacterium and cell line the l. casei atcc 39392 strain used in this study was deposited in atcc and is a plasmid-free strain grown in man-rogosa-sharpe (mrs) medium (sigma) at 37°c without shaking. the recombinant l. casei (pg:612-20mt6d) was generated in previous study (jiang et al. 2014) . chloramphenicol (cm) and kanamycin (sigma) were each used at a final concentration of 10 μg/ml. tgev was previously isolated and purified in our laboratory. swine testicle (st) cells were cultured in dulbecco's modified eagle's medium (dmem, gibco) supplemented with 10 % foetal bovine serum (fbs, gibco) at 37°c with 5 % co2. the virus stocks were clarified by centrifugation at 800×g for 10 min to remove cell debris, titrated using the cytopathic effect assay and then stored in aliquots at −80°c until needed. tgev-seronegative crossbreed (large white) piglets were obtained from a local breeding farm after birth. the piglets were housed separately in specialized cages that were maintained in sterile stainless steel isolators (five piglets/isolator) and fed commercial sterile milk and water. four groups (n = 5 each) of piglets were orally dosed with 10 10 cfu of pg:612-20mt6d in 1 ml of pbs or pbs alone (jiang et al. 2014) ; this formulation was used to immunize piglets via an intragastric route in a different immunization protocol. the first group was immunized with pg:612-20mt6d in 1 ml for priming. the second group was immunized with pg:612-20mt6d in 2 ml for priming. the third group was immunized with pg:612-20mt6d in 2 ml for 48 consecutive hours. the forth group was immunized with pg:612-20mt6d in 1 ml for 48 consecutive hours. the control group was immunized with pbs. the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. seven days after immunization, serum samples were prepared from collected blood samples. the intestinal mucus was collected by rectal swab and subsequently homogenized for 30 min in 400 μl of sterile pbs (ph 7.4) containing 0.01 mol/ l edta-na2 and then incubated for 12 h at 4°c. clear extracts of all samples were collected by centrifugation at 3000×g for 10 min and stored at −80°c with protease inhibitors for subsequent analysis. enzyme-linked immunosorbent assay (elisa) plates were coated for 18 and 12 h at 4°c with full tgev and the vp4 protein, respectively, which were previously isolated and purified in our laboratory. cultured st cells were used as a negative antigen control. after the wells were blocked for 12 h at 4°c with pbs containing 5 % skim milk, the plates were washed three times with pbs + tween 20 (0.1 %) (pbst). mucus and serum (diluted 1:50) samples were added to the wells in triplicate and incubated for 1 h at 37°c. afterwards, the plates were washed three times with pbst, and a horseradish peroxidase-conjugated goat anti-pig igg or iga antibody (invitrogen) was added to each well (1:5000) and incubated for an additional 1 h at 37°c. after another round of washing, colour development was accomplished using ophenylenediamine dihydrochloride as a substrate, and the absorbance was measured at 490 nm. naive purified spleen t cells (5 × 10 5 cells/ml) were cultured in 24-well tissue culture plates and stimulated with 1.0 μg ml −1 of plate-bound anti-cd4(pe) antibody (abcam) in complete rpmi 1640 medium. single-cell suspensions were stimulated in culture with ionomycin (2 μg ml −1 ) in the presence of monensin (2 μm) for 4-6 h of culture. the cells were surface labelled and then fixed, permeabilized and intracellularly labelled with ifn-γ and il-4 antibodies as previously described (moore-connors et al. 2013; zhou et al. 2009 ). for th1 and th2 differentiation, the cells were stimulated in the presence of 10 ng ml −1 anti-ifn (fitc) and 10 ng ml −1 anti-il-4 (fitc) (abcam) antibodies. the cells were labelled with carboxyfluorescein succinimidylester (cfse) according to a previous protocol (jiang et al. 2014) . the data were acquired by gating on the cd4 + cell population with a facscalibur cytometer. the sequential loss of cfse fluorescence was used to measure cell division and proliferation. groups were housed in the same facility but separated by room and ventilation system. pigs in each room were confined by pens on a solid floor that was rinsed daily, fed a balanced diet ad libitum based on weight and provided free access to water. tgev-challenged pigs received a 5 ml dose of 1 × 10 5 plaque-forming units (pfu)/ml via oral-gastric gavage on 0 days post-inoculation (dpi). pigs in the control group were administered volume-matched virus-free cell culture media. the control, immunized and no challenge group pigs were randomly selected for necropsy on the fourth day. to assess histological changes in the intestinal tissues, both the intestine and other major organs were examined at necropsy. after 48 h of fixation in 10 % neutral buffered formalin, tissue sections were trimmed, processed, and embedded in paraffin, sectioned, stained with haematoxylin and eosin (h&e) and then examined for pathological changes by light microscopy using a model microscope (olympus, tokyo, japan). real-time rt-pcr (qrt-pcr) was employed to determine the amount of tgev and cytokine gene products (isgs) in rectal swab samples and the intestinal tissues using a cfx96tm real-time pcr detection system (bio-rad). total rna was extracted from faecal samples and splenic and intestinal tissues using viral rna extraction and total rna extraction kits (intron) according to the manufacturer's instructions. the extracted rna was subjected to real-time qrt-pcr using a one-step sybr® qrt-pcr reagent kit (takara, shiga, japan). following reversetranscription of the viral rna at 45°c for 20 min, the resulting cdnas were used for real-time pcr amplification. a standard curve was generated by plotting threshold values against serially diluted plasmid dna encoding the fragment of the tgev spike protein (lee et al. 2011 ). all determinations were performed using data from wells evaluated in duplicate to ensure reproducibility. the copy number of the experimental samples was determined by interpolating the threshold cycle values using the standard curve. real-time quantitative pcr was utilized to quantify the products of interest (trl-2, −4, −9, il-4, il-17, ifn-γ and tgf-β) relative to the quantity of messenger rna (mrna) in the total rna isolated from the splenic and intestinal tissues (dirisala et al. 2013; kiros et al. 2011) ; the specific primers are listed in table 1 . the livak method (δδct method) was used to calculate the fold change compared to the β-actin gene control. gene expression data were expressed relative to unimmunized and uninfected piglets. to phenotype immune cells in the spleen and mesenteric lymph nodes, single-cell suspensions were isolated and labelled with fluorochrome-conjugated antibodies. to determine the cell type and the frequency of ifn-γ and il-4-producing th1 and th2 cells, single-cell suspensions were stimulated in culture with ionomycin (2 μg ml −1 ) in the presence of monensin (2 μm) for 4-6 h. the cells were surface labelled to detect cd4 and then fixed, permeabilized and intracellularly labelled with ifn-γ and il-4 antibodies as previously described (moore-connors et al. 2013; zhou et al. 2009 ). comparison of the piglets' iga and igg titres was conducted by a paired t test. the th cell, cytokine expression and faecal pedv rna shedding titres among litters were compared using one way analysis of variance (anova) followed by duncan's multiple range test. the mucosal immune response of the piglets was evaluated by measuring the iga response in diluted intestinal lavage fluid post-intragastric immunization. as shown in fig. 1a , the newborn piglets that received 2 ml of recombinant lactobacillus pg:612-20mt6d had the highest mucosal iga levels after immunization. the iga levels at 48 h in the piglets that received 4 ml were slightly lower than the newborn piglets that received 2 ml throughout the process. the vaccine doses also provoked systemic immunity based on the serum analysis and elicited specific igg responses from the immunized piglets (fig. 1b) . newborn piglets that received 2 ml of the vaccine also had the highest specific igg level. the specific igg titre reached as high as 1:1600. finally, the antibody kinetics in the serum and intestinal lavage samples from the animals showed that the specific antibody igg and iga levels were increased on the seventh and eighth days and the titre was decreased during the last week without immunization. to analyse the effect of recombinant lactobacillus pg: 612-20mt6d on t helper cells, we evaluated t helper cell polarization. throughout the process, we utilized the model of immunization described here. as shown in fig. 2 , the immune response balance mediated by th1 and th2 was broken in favour of th1-mediated immunity. the pg:612-20mt6d/l. casei group exhibited 80 % protection within 4 days of challenge with tgev (pg:612-20mt6d/l. casei) (fig. 3) . in contrast, the control group piglets immunized with pbs all died after tgev challenge. all piglets that died/were killed were emaciated and had yellow faeces coating the skin and hair. in some piglets, the intestinal lumens were filled with large amounts (approximately 50-70 ml) of yellowish foamy fluid. in other piglets, the walls of the small intestine were transparent and thin and the intestinal lumens were empty. no significant gross lesions were observed in other major organs (lung, kidney, liver and heart). formalin-fixed intestinal tissue sections from piglets treated with different treatment modalities were blindly analysed for histopathological changes associated with tgev infection. according to the histopathological analysis, the small intestine samples from the three groups (positive, negative and immunized groups) showed obvious differences. as indicated in fig. 4 , different degrees of pathological changes were detected after infection, especially in the positive group where serious damage was observed. the representative pathological changes included intestinal villi hyperaemia, atrophy and destruction. in the pbs infection group, the jejunum villi were severe atrophied and destroyed, and the ilea exhibited severe lymphocyte proliferation in the lamina propria. the recombinant lactobacillus group showed jejuna with intact villi but low-grade hyperaemia and lymphocyte proliferation, and the ilea exhibited lymphocyte proliferation in the lamina propria. both the pbs and vaccine groups had severe inflammatory responses. the negative control piglets that were not infected showed normal histology. the sequences of the two primers were checked using the ncbi blast software, and no significant alignment with any other animal virus gene was found piglets inoculated with virulent tgev shed the virus for 12 h, followed by profuse diarrhoea that led the piglets to the verge of death 1-4 days after inoculation. the tgev load shed in the diarrhoea was 3.2 × 10 5 copies at the 12th hour, peaked at 1.72 × 10 6 copies at the 48th hour and then decreased until death in the pbs group (fig. 5) . the recombinant lactobacillus vaccine group (pg:612-20mt6d) exhibited the same trend. the tgev copy number was 10 5 at the 12th hour, and the copies reached a peak at the 48th hour. the copy numbers were similar and followed the same trend after reaching the peak. however, the copy numbers in the pg:612-20mt6d group were significantly lower than in the pbs group. next, we investigated the generation of lactobacillus vaccineinduced regulatory cells after infection in piglets. as shown in immunized with pg:612-20mt6d and pbs piglets were orally challenged with tgev. tgev-challenged pigs received a 5-ml dose of 1 × 10 5 plaqueforming unit (pfu)/ml via oralgastric gavage on days postinoculation. pigs (control group) were administered volumematched virus-free st cell culture media. all piglets were euthanized at 5 days for necropsy examination fig. 6 , there was a marked increase in the production of il-4 in cd4+ t cells. the th1 immune response induced by the vaccine was seriously broken in favour of th2-mediated systemic and mucosal immunity post-infection. the systemic th2 immune response was higher than the mucosal th2mediated immune response. after tgev infection, the body activated more th2 to protect itself in response to the infection. as shown in fig. 7 , toll-like receptor (tlr) expression was detected in the splenic lymphocytes (sl) and mesenteric lymph node cells (ml) in the piglets in the pbs and vaccine groups. tlr-2 was higher in the vaccine group than in the pbs group. in contrast, there were no significant differences between tlr-4 and tlr-9. however, the three tlrs exhibited the same trends in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine. tgev infection induced tlr expression and especially enhanced tlr-2 and tlr-4 expression, but the expression levels in the lactobacillus vaccine group were significantly lower than the levels in the pbs group. cytokine expression in the splenic lymphocytes and mesenteric lymph node cells was also analysed and compared in our study. the ifn-γ, il-4 and il-17 levels in the splenic lymphocytes from the lactobacillus vaccine group showed marked changes, whereas no significant difference was observed in the level of tgf-β. tgev infection stimulated cytokine expression in the pbs group, including ifn-γ and il-4. the vaccine group did not express a notably higher level of ifn-γ and il-4 compared with the pbs group. the tgf-β expression level in the mesenteric lymph node cells was lower in the vaccine group than in the pbs group; the same trend was observed with ifn-γ and il-4. the vaccine group provoked higher il-17 expression than the pbs group following tgev infection. the il-17 expression levels in both the splenic lymphocytes probiotics are well known to have additive effects on human health in terms of improving the gut microflora and modulating immune responses (villena et al. 2006) . studies have also reported that probiotic feeding results in an increased spleen mass, followed by higher levels of total serum proteins, increased globulin levels and enhanced production of secretory iga (dock et al. 2004) . in humans, lactobacilli colonize the distal small bowel and the large intestine. different probiotic bacteria possess various mechanisms, including adhesins and/ or coaggregation factors, which aid in adhesion and colonization (friedrich 2013) . during this period, immunization with recombinant lactobacillus is crucially important on the effects of immunization, such as the timing of the first immunization in the protocol and the dose. from these results, we show that the immune response in response to the first priming immunization dose is better than the response to the second immunization because the priming dose is better at initiating adhesion and colonization in the piglet. interestingly, the mucins are large glycoproteins that are the major organic components of mucus. the mucin protein content of the mucus is 20 %, whereas the carbohydrates comprise 70 to 80 % by weight. intestinal mucin has been shown to inhibit the replication of rotavirus in vitro (chen et al. 1993; superti et al. 1993 ). additionally, a high dose of lactobacillus adversely affects the immunization. there is some evidence of diarrhoea after a double dose of the lactobacillus vaccine, but from this result, we find that the two-dose immunization is still the best immunization plan. the reason for the diarrhoea after immunization is the overdose of lactobacillus, which is a type of microbe that causes a disturbance in the intestinal microbial flora for short time. many enteric pathogens must first adhere to the intestinal epithelial cells to initiate intestinal disease. limiting access of the pathogens to intestinal epithelial cells is one strategy to prevent disease that has been investigated previously. for example, the competitive inhibition of bacterial adherence by mimicry of receptors on the apical surface of enterocytes using oral administration of sialylated glycoproteins has been shown to protect newborn calves from the enterotoxigenic e. coli strain k99 (mouricout et al. 1990 ). lactobacillus must colonize the gut soon after birth; therefore, the vaccine could play a role in non-specific immunity. probiotic strains with a high adherence capacity have been demonstrated to enhance the immunoglobulin a response to rotavirus (kaila et al. 1992) . in this study, we observed a significant increase in the anti-tgev iga titre in the intestinal tract of piglets administered recombinant l. casei. furthermore, we showed that the diarrhoea state of piglets administered l. casei was significantly lower than that of piglets administered saline. in the murine model of ifv infection, the virus moves from the upper respiratory tract to the lower respiratory tract (hori et al. 2001) . hrv-vaccinated and lactobacillus acidophilus-fed pigs had a significantly higher magnitude of hrv-specific iga and igg antibody-secreting cell responses in the ileum and serum igg antibody and virus neutralizing antibody titres compared to hrv-vaccinated pigs without l. acidophilus colonization (zhang et al. 2008) . our immunization stimulated the same systemic specific igg titres. the specific antibody response neutralized the challenged tgev, and the load of tgev in the diarrhoea was significantly decreased. the mucosal immune response formed the first barrier function to neutralize tgev. in large scale swine farm surveillance, lower piglet birth weight and higher within-litter variability in birth weight were factors associated with higher losses from birth to weaning (yuan et al. 2015) . during tgev infection, it is likely that the stronger piglets obtained more iga than their non-immunized counterparts and thus were more likely to survive until the intestinal villi regenerated and immunity developed. during the histopathological analysis, some damage was observed in the small intestine. for instance, the villus wall of the control groups was thin and almost transparent, probably due to atrophy. lymphocyte proliferation in the intestinal lamina propria was also found in some piglets administered an oral dose of recombinant lactobacillus, indicating that recombinant lactobacillus induced a mucosal immune responses in the piglets. taken together, our data show the tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev infection with intestinal tropism in piglets. we evaluated the specific t cell immune responses induced by the recombinant l. casei vaccine compared with the pbs control group in the piglets. we demonstrated that l. casei significantly enhanced the immunogenicity of the tgev vaccine as indicated by the significantly higher magnitude of specific ifn-γ-producing cd4+ t cell responses. there has reported that mice fed recombinant l. casei with the adjuvant mdp and tuftsin have significantly higher th1 and th17 production than control group mice (jiang et al. 2014) . these results were the same and indicated that l. casei had a strong potentiating effect on both the cellular and humoral immunity induced by the oral l. casei vaccine. similarly, a previous study showed that oral intake of l. fermentum cect5716 fig. 7 cytokines and tlr expression. the rna of splenic lymphocyte (sl) and mesenteric lymphocyte (ml) in immunized piglets, pg:612-20mt6d and pbs groups, were used to analysed the cytokines and tlr expression. ifn-γ, il-4, il-17, tgf-β, tlrs expressing were detected in splenic lymphocyte (sl) and mesenteric lymph cells (ml) piglets, such as pbs and vaccine groups. *significant difference by student t test (p < 0.05). data shown were compared using one-way analysis of variance (anova) followed by duncan's multiple range test. representative for three independent experiments significantly enhanced serum th1 type cytokine production and influenza-specific iga antibody responses to an intramuscular influenza vaccine in adults (olivares et al. 2007) . the mesenteric lymph nodes were primarily used to analyse tgev infection and the immunoprotection provided by the lactobacillus vaccine in terms of mucosal immunization and infection. ifn-γ induction by tgev results from interactions between an outer membrane domain of el and the pbmc membrane (charley and laude 1988) ; however, these authors did not study the expression of il-4 by pbmcs. the expression of ifn-γ was higher than il-4 in the immunized group, and the th1/th2 balance was broken in our study. after immunization with recombinant lactobacillus, ifn-γ played a major role in the mucosal immune response. however, after tgev infection, the systemic and local immune responses shifted from th1 to th2. the systemic humoral immune response was stronger than the cellular immune response after tgev infection. this is the first study to demonstrate that tgev infection polarized the immune response to th2 immunity and that recombinant lactobacillus could weaken tgev infection in the form of th2 immunity. from these results, we found that the immunization did not polarize th2 immunity more seriously compared to the pbs control group. the proteinbased sars coronavirus vaccine boost induced similar levels of th1 and neutralizing antibody responses that protected vaccinated mice from subsequent sars-cov challenges but induced stronger th2 and ctl responses (zheng et al. 2008) . the uv-inactivated sars coronavirus vaccine retained its immunogenicity and promoted th2type immune responses (tsunetsugu-yokota et al. 2007 ). the activation of th2 responses such as il-10 stimulate b cell proliferation, which can produce specific and nonspecific anti-infection antibodies (grodeland et al. 2015) ; similarly, both t and b cells have functions following lactobacillus vaccination. the production of il-9 by th2 cells results in the proliferation of mast cell growth, and il-13 stimulates epithelial cell growth (tukler henriksson et al. 2015) . the proliferation of epithelial cells is crucial for tgev infection. the th2 response could also stimulate the production of mucus by epithelial cells (zhang et al. 2015) . il-17 cells play an essential role in mhv-induced immunopathology, and ifn-γ is important for maintaining the immune balance between th1 and th17 responses during acute viral infection (yang et al. 2011 ). however, the th1/th2 balance in the negative control group was different than the balance in the immunized group. tgev affects both systemic and local cellular immunity without immunization. il17 has been reported to have both pro-and anti-inflammatory effects (lafdil et al. 2009; nagata et al. 2008) . our results showed that the immunized piglets provoked il-17 expression form both the systematic and mucosal immune responses after tgev infection. compared with the mucosal immune response, il-17 expression in the mesenteric lymph nodes was markedly lower than the expression in the spleen cells. however, il-17 expression in the pbs group was lower than the expression level in the immunized groups, indicating that the lactobacillus vaccine group activated il-17 expression during tgev infection. swine-origin influenza a virus-infected patients exhibited rapid lymphopenia, t cell activation and a preferential loss of the th17 subset during the early stage of acute infection (jiang et al. 2010) , which was consistent with the first reports that swine-origin virus inhibited th17 proliferation after infection. our study also found the same phenomenon after coronavirus infection in swine compared with the immunized group. the most important finding was that the oral recombinant lactobacillus vaccine stimulated th17 cell proliferation. the proliferation was involved in cytokine and chemokine production, neutrophil recruitment, promotion of t cell priming and antibody production (dirisala et al. 2013) . th17 responses are protective against lethal influenza virus infection in il-10deficient mice (mckinstry et al. 2009 ). in contrast, a deleterious role of il-17 has been proposed to contribute to the acute lung injury associated with il-17-mediated neutrophil recruitment during influenza virus infection (crowe et al. 2009 ). there were significant changes in the il-4 and ifn-γ expression levels in the mesenteric lymph node cells compared with the pbs group. moreover, il-4 expression was higher than ifn-γ expression in the spleen cells. the expression of il-4 and ifn-γ indicated that the recombinant lactobacillus effectively inhibited inflammation after tgev infection. in this study, we also evaluated tlr-2, tlr-4 and tlr-9 expression in piglets immunized with recombinant l. casei and then challenged with tgev. previously, tlr expression in pigs has been studied only at the mrna level in lymphocytes using real-time pcr because antibodies against porcine tlrs are not currently available. tgev infection did not induce tlr-2, tlr-4 and tlr-9 expression in the spleen cells. however, tlr expression was significantly different in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine groups. tlr expression was extremely high in the pbs group compared with the other groups. the cytokine and tlr expression levels in the splenic lymphocytes are indicators of systemic immunity, whereas the expression in the mesenteric lymph node cells was associated with the local and mucosal immune responses. the expression levels of all tlrs in mesenteric lymph node cells were higher in the pbs group than the vaccine group, suggesting that the lactobacillus vaccine effectively inhibited tlr expression. the recombinant lactobacillus groups exhibited jejuna and ilea with lymphocyte proliferation in the lamina propria. transmissible gastroenteritis (tge) coronavirus infection resulted in antibody production from primed mesenteric lymph node cells following an in vitro boost with viral antigen (berthon et al. 1990 ); as an intestinal infectious disease, tgev would attack the intestinal tissue and local immune system. exposure of pigs to tgev or prcv results in distinct disease patterns related to differences in tissue tropism (saif 1996) . however, the increased frequencies of tlr-2 and tlr-9 expression in pigs may simply be due to the significantly higher counts of l. casei or mdp and tuftsin, which may translate to an increased magnitude of tlr agonists available to stimulate the host mucosal immune system. it is likely that the higher lab count in the lab plus hrv group played a more pertinent role in the significant increases in tlr2 and tlr9 expression (wen et al. 2009 ). taken together, tgev immune protection was primarily dependent on the mucosal immune response. systemic immunity did not play a key role after tgev infection. interestingly, il-17 expression in the vaccine group was significant higher than il-17 expression in the pbs group challenged with tgev, and th17 played an anti-inflammatory role in mucosal immunity. il-17 also stimulated intestinal epithelial cell differentiation and growth. in conclusion, our study suggests that the recombinant lactobacillus vaccine provokes specific mucosal and systemic immune responses to protect piglets from infection. iga played a dominant role in the mucosal immune response after tgev challenge. therefore, the histopathology and rna copy numbers directly demonstrated that the lactobacillus vaccine was effective for tgev infection. the protective efficacy was significantly higher, which would have great value in practice. moreover, although the recombinant lactobacillus vaccine-induced th1 immunity, the immune balance was broken by tgev infection; thus, the immunized piglets initiated a th2 immune response against tgev infection. taken together, we showed that il-17 signalling was vital for lactobacillus vaccine immunized piglets infected with tgev. the lactobacillus vaccine also inhibited tlr expression, which indicated that most of virus was neutralized because the tlrs were not activated by the virus. a transmissible gastroenteritis coronavirus nucleoprotein epitope elicits t helper cells that collaborate in the in vitro antibody synthesis to the three major structural viral proteins cooperation between transmissible gastroenteritis coronavirus (tgev) structural proteins in the in vitro induction of virus-specific antibodies bioecological and nutritional control of disease: prebiotics, probiotics and synbiotics kinetics of the in vitro antibody response to transmissible gastroenteritis 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interest the authors declare that they have no competing interests.ethical statement the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. this article does not contain any studies with human participants performed by any of the authors. key: cord-348310-nc1tq5af authors: vázquez-ramírez, daniel; jordan, ingo; sandig, volker; genzel, yvonne; reichl, udo title: high titer mva and influenza a virus production using a hybrid fed-batch/perfusion strategy with an atf system date: 2019-02-23 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-09694-2 sha: doc_id: 348310 cord_uid: nc1tq5af a cultivation strategy to increase the productivity of modified vaccinia ankara (mva) virus in high-cell density processes is presented. based on an approach developed in shake flask cultures, this strategy was established in benchtop bioreactors, comprising the growth of suspension age1.cr.pix cells to high cell densities in a chemically defined medium before infection with the mva-cr19 virus strain. first, a perfusion regime was established to optimize the cell growth phase. second, a fed-batch regime was chosen for the initial infection phase to facilitate virus uptake and cell-to-cell spreading. afterwards, a switch to perfusion enabled the continuous supply of nutrients for the late stages of virus propagation. with maximum infectious titers of 1.0 × 10(10) iu/ml, this hybrid fed-batch/perfusion strategy increased product titers by almost one order of magnitude compared to conventional batch cultivations. finally, this strategy was also applied to the production of influenza a/pr/8/34 (h1n1) virus considered for manufacturing of inactivated vaccines. using the same culture system, a total number of 3.8 × 10(10) virions/ml was achieved. overall, comparable or even higher cell-specific virus yields and volumetric productivities were obtained using the same cultivation systems as for the conventional batch cultivations. in addition, most viral particles were found in the culture supernatant, which can simplify further downstream operations, in particular for mva viruses. considering the current availability of well-described perfusion/cell retention technologies, the present strategy may contribute to the development of new approaches for viral vaccine production. electronic supplementary material: the online version of this article (10.1007/s00253-019-09694-2) contains supplementary material, which is available to authorized users. modified vaccinia ankara (mva) virus is a highly attenuated poxvirus with promising properties as a vectored vaccine. mva initiates but cannot complete a full replication cycle in human recipients and is therefore immunogenic similar to live virus vaccines (gomez et al. 2013 ) but with safety properties resembling inactivated virus vaccines (gilbert et al. 2006; cebere et al. 2006; webster et al. 2005; stickl et al. 1974; mayr 2003) . mva recombinants expressing different viral heterologous antigens have been tested in pre-clinical and clinical trials as candidate vaccines against infectious diseases such as aids, influenza, severe acute respiratory syndrome (sars), and human respiratory syncytial virus (rsv) infection (boukhebza et al. 2012; gilbert 2013; gomez et al. 2011 gomez et al. , 2012 . conventionally, mva seed virus stocks considered for vaccine manufacturing are produced in chicken embryo fibroblast (cef) that are fully permissive for mva. using wellestablished protocols (altenburg et al. 2014; cotter et al. 2017) , large-scale production of mva recombinants for immunization campaigns would also have to rely on propagation in cef (altenburg et al. 2014 ). however, because supply with the primary cell cultures can be challenging for large scale manufacturing, production of mva recombinants is being investigated for continuous suspension cell lines, such as the duck cell lines age1.cr and age1.cr.pix lohr et al. 2009 ), or the duck embryonic stem cellderived eb66 cells (léon et al. 2016) . influenza viruses are usually processed to provide inactivated vaccines against seasonal epidemics (soema et al. 2015) . production of influenza vaccines in the past 70 years relied on embryonated chicken eggs. disadvantages associated with this substrate are the use of an animal-derived substrate and the potential shortage of eggs especially in case of a pandemic emergency. recently, two cell culture-based vaccines produced either in mdck cells or in insect cells using the baculovirus expression vector system were approved by the fda (buckland 2015) . in addition, efficient production of influenza virus has also been shown for other suspension cell lines including vero cells (litwin 1992; paillet et al. 2009 ), per.c6 cells (pau et al. 2001) , hek293 cells (le ru et al. 2010) , eb66 cells (brown and mehtali 2010; white et al. 2018) , and age1.cr as well as age1.cr.pix cells (jordan et al. 2013; lohr et al. 2012; lohr 2014) . cell culture-derived viral vaccines are typically produced in biphasic processes. they comprise an initial cell growth phase and a virus replication phase that initiates with inoculation by seed virus. after the viral genome is amplified and viral proteins are produced, virions are assembled and progeny virus particles released (aunins 2000) . typically, cells are first cultivated in batch mode and infected in the late exponential growth phase at concentrations in the order of 10 5 -10 6 cells/ml, with or without a partial exchange of culture medium (aunins 2000; tapia et al. 2016) . titers of wild-type mva obtained in adherent cultures of cef cells in serum-containing medium are in the range of 10 7 -10 9 infectious units (iu) per ml (gilbert et al. 2005; meiser et al. 2003 ). the avian cell lines age1.cr.pix lohr et al. 2009; lohr 2014) , eb14 (guehenneux and pain 2005) and eb66 (léon et al. 2016 ) also yield titers in the order of 10 8 iu/ml but with the advantage of enabling suspension cell culture processes. however, one caveat is that these processes require induction of suspended cell aggregates for efficient replication of mva. a novel mva derivative, mva-cr19, was adapted to propagation in true single-cell suspension cultures without the requirement for addition of medium to induce cell aggregation (jordan et al. 2013) . scalable and intensified processes that yield high titers are desirable to secure adequate supply with vaccines. in the case of mva, high virus titers are required because mva does not replicate in human recipients and therefore is not amplified at the site of injection. concentrated doses of 10 8 -10 9 iu/ml are estimated to be required for clinical applications of mva (gomez et al. 2013; altenburg et al. 2014 ). in the case of influenza vaccines, intensification is desirable because composition of multivalent vaccines changes every year, and the time is short between selection of seasonal virus strains and desired start of vaccination. increasing the concentration of host cells is a standard approach to intensify production processes for biologicals. processes at high cell density (hcd) allow the use of compact bioreactors with high volumetric production rates and can be adjusted to viable cell densities of 10 7 -10 8 per ml (clincke et al. 2013) . such cell densities can be achieved in perfusion mode, which allows for a continuous addition of fresh medium and removal of toxic by-products (such as lactate and ammonium) while retaining cells in the bioreactor using different retention systems. the production of recombinant proteins in perfusion is typically performed at high-medium exchange rates of 1-3 media volumes per reactor volume per day (1/day) or 0.05-0.5 nl/(cell × day) (konstantinov et al. 2006 ). to maintain cultures in a proliferative state at constant high cell densities, a controlled and continuous removal of cells from the bioreactor is performed, the so-called bcell bleed ( clincke et al. 2013; deschênes et al. 2006; hiller et al. 1993) . novel cultivation strategies such as n-1 perfusion/high-seed fed-batch (yang et al. 2014) , concentrated fed-batch (fb) (yang et al. 2016) , and hybrid perfusion fb (hiller et al. 2017) have been developed to minimize media use while maintaining cell-specific and volumetric productivities as well as guaranteeing consistent product quality. in the field of viral vaccine production, high cell concentrations up to 5 × 10 7 cells/ml were used for the propagation of influenza a/pr/8/34 (h1n1) virus (genzel et al. 2014 ) and the mva-cr19 virus strain (vazquez-ramirez et al. 2018 ). at those cell concentrations, virus propagation must be performed at optimal ph, temperature, and nutrient concentrations to avoid the so-called bcell density effect^, a reduction in cell-specific virus yield often observed for concentrations exceeding 5 × 10 6 cells/ml (lindsay and betenbaugh 1992; maranga et al. 2003) . several improvements were reported for the hcd production of influenza a/pr/8/34 (h1n1) virus. cultivations were performed in perfusion cultures for both cell and virus propagation phases using an acoustic filter (petiot et al. 2011) or an alternating tangential flow (atf) system for cell retention (genzel et al. 2014) . the perfusion systems differed in the partitioning of the virus. whereas the acoustic filter allowed a continuous virus harvest, atf systems with membrane pores sizes < 0.5 μm retained most released viral particles in the bioreactor. in addition, the continuous permeate flow through the hollow fiber membrane resulted in product losses due to unspecific binding or membrane fouling by entrapment of cellular debris and virus particles within the membrane pores (genzel et al. 2014) . although cell-specific virus yields comparable to conventional batch processes were obtained for both cell retention systems, the volumetric productivity (the amount of virus per volume of medium spent and time) was lower (genzel et al. 2014 ). this strategy would therefore be less competitive because of the increased cost of goods (cogs) for its implementation in large-scale (pollock et al. 2013) . in an attempt to produce mva virus (mva-cr19 strain) at high concentrations in age1.cr.pix cells (5 × 10 7 cells/ml), perfusion using a medium free of animal-derived components was implemented (vazquez-ramirez et al. 2018 ). this method resulted in virus retention and yielded lower cellspecific and volumetric productivities compared to conventional cell density cultivations. a detailed analysis of several cultivation strategies in shake flasks demonstrated that a fb phase followed by a daily medium exchange of 90% can result in an improvement of both cell-specific yield and volumetric productivity, even surpassing conventional cell density processes performed as a control (vazquez-ramirez et al. 2018) . while shake flask experiments are essential for the characterization of production processes, they are insufficient for establishing strategies for large-scale manufacturing of vaccines. hence, in the present study, an optimized hcd process was developed in a controlled and scalable cultivation system to provide the required high yields of mva virus. hcd cultures were achieved in a 1 l bioreactor with an atf perfusion system using a manual perfusion control. perfusion rates were adjusted applying a fixed cell-specific perfusion rate (cspr), which is the volume of medium provided to a single cell per day (ozturk 1996) . during virus production, the cells were cultivated in fb followed by a continuous medium exchange with the same atf perfusion system used for the cell proliferation phase (hybrid fb/perfusion strategy). yields and productivity of this process confirmed shake flask results. to investigate options for the use of this strategy to other viral vaccine production processes, propagation of influenza a virus was also tested. again, a similar or even higher productivity compared to conventional cell density cultivations was obtained. the age1.cr.pix cell line (here named cr.pix) is directly derived from the avian cell line age1.cr, which was generated from muscovy duck retina cells ). the cr.pix cells differ from their progenitor age1.cr in that they express the pix protein of human adenovirus ), a virus that is not related to mva or influenza viruses. suspension cr.pix cells were cultivated in chemically defined cd-u3 medium (biochrom gmbh) with a glucose concentration of 33-40 mm, supplemented with glutamine (sigma, lot slbs8600) and alanine (sigma, lot bcbs2461v) to a final concentration of 2 mm. in addition, recombinant insulin-like growth factor (long-r3igf, sigma, lot los6008) was added at 10 ng/ml final concentration. cells were passaged every 3-4 days at a seed concentration of 0.8 × 10 6 cells/ml. cr.pix cells were inoculated in a 1 l (nominal volume) benchtop bioreactor (biostat®b plus, sartorius ag) at 0.8 × 10 6 cells/ml in a working volume (v w ) of 0.6-0.8 l (table 1) . bioreactors were operated at 37°c, ph 7.2, and a stirring speed of 120-160 rpm. dissolved oxygen concentration (do) was controlled at 40% by pulsed aeration with pure oxygen through a 20-μm pore size micro-sparger unit to a maximum of 29-38 cm 3 /min. cells were initially cultivated in batch until a glucose concentration of 14-17 mm (60-72 h after inoculation) was reached. at that point, perfusion was started using an atf2 perfusion system controlled by the c24u-v2.0 controller from refine technology and polysulfone hollow fiber cartridges with pore sizes of 500 kda (ufp-500-e-4x2ma, ge healthcare) and 0.65 μm (cfp-6-d-4x2ma, ge healthcare), or polyethersulfone hollow fiber cartridges of 0.2 μm (s06-p20u-10-s, spectrum labs) ( table 1) . cell suspension flow rate within the hollow fiber was set at 1.0 l/min, and defined perfusion rates were applied to achieve cell densities > 25 × 10 6 cells/ ml. perfusion flow rates during the cell growth phase were adjusted manually every 12 or 24 h. for that, viable cell densities were measured off-line, and the corresponding flow rates for that sampling time were calculated to assure a cspr of 0.06 nl/(cell × day), which is the optimal exchange rate for cr.pix cells based on their glucose consumption rate (vazquez-ramirez et al. 2018 ). expected viable cell densities and the corresponding perfusion flow rates after 12 or 24 h were calculated taking into account a maximum cell-specific growth rate of μ = 0.026 h −1 (data not shown). a linear profile between sampling time points was achieved using the cascade control of the biostat®b plus module. two hours before infection, one reactor volume was exchanged with fresh medium using the atf system. a summary of key parameters of all perfusion cultivations is presented in table 1 . after medium exchange, bioreactors were infected either with mva-cr19 or influenza a virus a/pr/8/34 (h1n1). for mva-cr19 virus, two bolus feeding regimes followed by a perfusion regime, namely hybrid 1 (fig. 1a) and hybrid 2 ( fig. 1d) , were applied to optimize virus propagation at high cell densities. for hybrid 1, one-half of the cell suspension was discarded, and the virus production phase was started with 0.3 l of cell suspension to allow for a fb with a volume expansion up to threefold higher than its initial v w , as described before (vazquez-ramirez et al. 2018) . the fb was started immediately after virus infection with the addition of 0.15 l of fresh medium to obtain the minimum culture volume at which the bioreactor impeller was at least 2 cm below the medium surface. the fb continued with bolus feedings of 0.15 l at 12 h post infection (hpi) and 0.3 l at 24 hpi. finally, perfusion was performed at a rate of one reactor volume per day 36-120 hpi (fig. 1a) . mva-cr19 virus particles (200-400 nm nominal size) were harvested using the same atf hollow fiber module (0.65 μm pore size) as in the cell growth phase. similarly, for hybrid 2, one bolus feed of 0.2 l was done immediately after virus infection followed by a second feed of 0.25 l at 24 hpi. subsequently, perfusion was again performed at a rate of one reactor volume per day 36-120 hpi (fig. 1d ). in order to improve the virus harvest, the 0.2-μm atf module used during the cell growth phase was replaced by a new 0.65-μm module at 36 hpi. for influenza a virus production, a hybrid strategy ( fig. 3a ) similar to the one assessed for mva-cr19 virus was established. reference processes, one operated completely in perfusion with total virus retention and one in batch at conventional cell densities, were performed as proposed previously by genzel et al. (2014) . a detailed description of the process parameters during cell expansion (table 1) and performance during virus propagation (table 2) is presented in the bresults^section. all infections with mva-cr19 virus were carried out with the working bank no. 22.08.2013 (4.41 × 10 8 infectious units per ml (iu/ml)) derived from a virus seed (jordan et al. 2013) kindly provided by probiogen ag. mva-cr19 virus seed aliquots were treated for 1 min in a sonication water bath to break up virus aggregates, diluted in fresh medium with a volume equal to 5-6% of the bioreactor v w , and added to the cell culture to an moi (multiplicity of infection) of 0.05 iu/cell. for all experiments with mva-cr19 virus, the infectious titers were determined taking into account its potential application as a (live) viral vector (jordan et al. 2013) . vaccinia viruses usually replicate in a highly cell-associated fashion. therefore, for the quantification of total virus titers (intraand extracellular), the cell suspensions were treated for cell lysis. the lysates were obtained by three freeze/thaw cycles (− 80°c/rt) followed by 1 min in a sonication water bath (45 khz). cellular debris was removed by centrifugation at 1500×g at room temperature for 10 min. for the quantification of virus released by host cells into supernatant, the samples were centrifuged at 200×g at rt for 5 min. the cell-free supernatant was also subjected to three freeze/thaw cycles before storage (jordan et al. 2013) . all virus samples were stored in aliquots of 0.5-1 ml at − 80°c. the number of infectious units was determined as described previously by jordan et al. (2009) with a relative standard deviation of ± 0.4 log. the resulting titers are expressed as iu/ml. the studies with human influenza a virus were performed with mdck-derived virus seed a/pr/8/34 h1n1 (robert koch institute, amp. 3138) that was adapted to cr.pix cells after three passages. the infectious titer of the adapted virus seed was determined by a tcid 50 assay as 1.48 × 10 7 iu/ml. all bioreactor experiments were performed at an moi of 1 × 10 −3 in the presence of 1 × 10 −6 u trypsin/cell (gibco, no. 27250-018; prepared in pbs to 500 u/ml) to facilitate progress of infection. as opposed to mva, the main application for influenza virus preparations is inactivated vaccine where the total concentration of the viral hemagglutinin protein as an antigen is decisive. for this reason, total virus particle concentrations were estimated by a hemagglutination (ha) assay as previously described by kalbfuss et al. (2008) . ha titers, expressed as log ha units per test volume (log hau/ 0.1 ml), were converted to virions/ml assuming the binding of one virus particle per erythrocyte and an erythrocyte concentration of 2 × 10 7 cells/ml, by: with c virus as the total virus concentration in virions/ml. the relative standard deviation of the method was ± 0.09 log (ha units/0.1 μl) (kalbfuss et al. 2008 ). the cell-specific virus yield (y v/cell ) was calculated based on the total (i.e., intra-and extracellular) number of infectious virus particles (vir t , infectivity assay) for mva virus and on the total number of all virus particles (c virus , ha assay) for influenza a virus, taking into account the total number of viable cells at the time of sampling (cell t ). the latter differed similarly, the volumetric productivity (p v ) was calculated considering vir t , the total spent medium during cell growth and virus replication phase (v t, l), and the total process time (t t, day), by: samples of 6-8 ml from bioreactor cultures were taken with a syringe through a luer-lock-septum in 12 or 24-h intervals and stored at − 80°c until analysis. a validated assay using a bioprofile 100 plus (nova biomedical) was used to determine glucose and lactate concentrations as described previously (lohr et al. 2009 ). viable cell density (vcd, cells/ml), cell viability (%), and average cell diameter (μm) were determined with the cell counter vi-cell™ xr (beckman coulter) using a previously validated measuring program with a relative standard deviation of 2.5% for age.cr and cr.pix cells (lohr 2014) . cells analyzed from a total of 100 images were clustered in diameter classes in the range of 8.1-29.9 μm for calculation of the total viable cell volume per culture volume (vcv, μl/ml) using the cell number and cell diameter distribution. for some cultivations, an incyte® capacitance probe connected to an arc view 265 controller (hamilton bonaduz ag) was evaluated for its performance to deliver on-line vcv data. the on-line system was configured to provide the cell culture's permittivity (ε, pf/cm), which correlates directly to the vcv. on-line permittivity was converted to vcvapplying a correlation factor vcv/ε of 1.8 obtained from previous cultivations (data not shown). a strategy previously reported for production of mva-cr19 virus at high cell densities in shake flasks (vazquez-ramirez et al. 2018 ) was transferred to a controlled stirred tank bioreactor with an atf2 system for cell retention. the method transfer was investigated for production of mva and influenza a virus. for the mva-cr19 virus, this process was adapted for its implementation in a 0.6-l (v w ) bioreactor. during the cell growth phase of variant bhybrid 1^, an actual cspr of 0.068 nl/(cell × day) ( fig. 1a and table 1 ) was obtained, which was comparable to the target 0.06 nl/(cell × day). this perfusion rate enabled a cell expansion from 1 to > 50 × 10 6 cells/ml (fig. 1b) with a μ mean of 0.021 1/h (table 1) . this was comparable to the assumed specific growth rate of 0.026 1/h for perfusion and was within the range of 0.016-0.023 1/h from previous reports for batch cultures (lohr et al. 2012) . manual control of the cspr during the cell growth phase also prevented any glucose limitation and the excessive accumulation of lactate (fig. 1c) . as an alternative to the hybrid 1, the hybrid 2 was started at 8 × 10 6 cells/ml and cultivated up to 26 × 10 6 cells/ml. the resulting cspr and μ mean were in average 0.057 nl/ (cell × day) and 0.024 1/h, respectively (table 1 ). these were also in accordance with the target values of 0.06 nl/(cell × day) and 0.026 1/h. in contrast to the hybrid 1 cultivation, the cell suspension was concentrated twofold before infection by reducing the working volume to 0.3 l (fig. 1d) , and one reactor volume was exchanged with fresh medium. this procedure shortened the cell growth phase to 2 days and obviated discarding half of the volume of the produced cell suspension (fig. 1e) . afterwards, the already described hybrid strategy was applied. the on-line vcv monitoring implemented in hybrid 2 correlated well with off-line measurements up to late stages of the mva virus propagation phase (fig. 1e) . this demonstrated the robustness of on-line capacitance measurements when the vcd exceeds 40 × 10 6 cells/ml, and expanded measurement to stages where virus-induced cell damage and apoptosis are widely spread within the infected cell population. in general, both the manually controlled cspr during the cell growth and the hybrid strategy applied during virus propagation prevented the glucose limitation and the excessive accumulation of lactate (fig. 1f) . a maximum mva-cr19 virus yield of 1.0 × 10 10 iu/ml was obtained at 96 hpi and 72 hpi for hybrid 1 and hybrid 2, respectively (fig. 2) . for hybrid 1, the virus particles were harvested using the same atf hollow fiber module (0.65 μm pore size) as in the cell growth phase, whereas for hybrid 2 a 0.2-μm module was used during the cell growth phase and replaced by a new 0.65-μm module for perfusion and virus harvest at 36 hpi. the relatively low virus harvest observed in hybrid 1 (fig. 2a) suggested that membrane fouling might have occurred during the cell growth phase. nevertheless, a similar maximum virus titer, cell-specific yield, and volumetric productivity were obtained compared to the reference process in shake flasks (table 2, f + d), which involved daily harvesting of virus (vazquez-ramirez et al. 2018) . even more significant is the more than tenfold increase in both the cellspecific yield and the volumetric productivity of hybrid 1 compared to the use of only perfusion during virus propagation (table 2 , perfusion) (vazquez-ramirez et al. 2018) . given the suboptimal virus harvesting for hybrid 1, the product collected in the permeate was not considered for the calculation of the cell-specific yield and the volumetric productivity. for hybrid 2, almost all infectious virions were found in the culture supernatant with a maximum virus titer of 1 × 10 10 iu/ml (72 hpi, fig. 2b ). this is in agreement with previous reports (jordan et al. 2013; vazquez-ramirez et al. 2018) . changing the hollow fiber module during the virus production phase enabled a quantitative harvest of virus particles from the supernatant only during the first 12 h of perfusion (i.e., 36-48 hpi) (fig. 2b) . from 48 hpi, the virus particle concentration in the permeate decreased considerably with respect to the apparent virus content in the culture supernatant. the virus titer in the permeate reached 1 × 10 6 iu/ml at 72 hpi, when the maximum virus titer of 1 × 10 10 iu/ml was reached in the culture supernatant (fig. 2b) . a further decrease in the permeate virus concentration to 1 × 10 5 iu/ ml was observed up to 120 hpi, while the titer in the bioreactor remained almost stable in the order of 10 9 iu/ml (fig. 2b) . similar to the hybrid 1 cultivation, these observations suggest significant membrane fouling at 48 hpi (24 h after begin of perfusion), preceding the most productive period of the virus propagation phase that occurs 48-72 hpi (fig. 2b) . this phase also coincides with a fast drop of cell viability and, very likely, with increasing in cell lysis. the resulting massive accumulation of cell debris and the aggregation of mva-cr19 viral particles (200-400 nm) may eventually have led to the obstruction of the atf membrane (0.65 μm nominal pore size). maximum titers were reached 72 hpi, and infectious virions appeared to remain stable in the supernatants and as well in the total cell lysates, as no obvious decay in titers was observed up to 120 hpi (fig. 2b) . despite the differences in the feeding profile during the fb, the hybrid 2 variant was as productive as hybrid 1 (table 2 ). in particular, a cell-specific virus yield of 352 iu/cell and a volumetric productivity of 2.8 × 10 11 iu/(l × day) were obtained. similar to the hybrid 1 variant, a very low amount of product was collected in the permeate line and, therefore, was neglected for the calculations of the cell-specific yield and the volumetric productivity. the reduced process time of hybrid 2 (8.2 days) led to a twofold higher volumetric productivity compared to cultivation hybrid 1. however, virus yields in the order of 10 11 iu/(l × day) would still be expected for a process operated at a similar time period as cultivation hybrid 1 (10.9 days). as the cell-specific virus yields were comparable to hybrid 1 (table 2) , the use of both strategies seems reasonable. an earlier study that investigated factors interfering with the production of influenza a virus at hcd gave only partial solutions regarding productivity optimization (genzel et al. 2014) . perfusion bioreactors were optimized in this earlier study with the avian cell line age1.cr in such a way that the cell-specific virus yields increased. however, overall volumetric productivity of these processes was higher in batch cultivations performed at conventional cell densities. the age1.cr cell line is the parental cell line of cr.pix cells and has shown higher productivities for influenza a virus. in contrast, the productivity was slightly lower for mva virus compared to cr.pix cells lohr et al. 2009 ). accordingly, it was investigated next whether the hybrid fb/perfusion strategy established for mva virus in this study could also be used for improving the volumetric productivity of influenza a/pr/8/34 (h1n1) virus in cr.pix cells at hcd. in addition, an alternative perfusion control was evaluated for the cell growth phase based on the glucose consumption, lactate accumulation, and the resulting medium acidification. to accomplish this, harvest and medium feeding pumps were activated when ph dropped to values below 7.2 and turned off when the ph increased to 7.2. under this condition, glucose is replenished at the same time that lactate is removed from the bioreactor. this perfusion control allowed for an average cspr of 0.119 nl/(cell × day) (table 1, fig. 3a ) and cell viabilities above 92% (fig. 3b) . additionally, it enabled to maintain glucose concentrations above 10 mm (fig. 3c ), similar to a previous report in shake flasks, where the ph was controlled in a range of 7.2 ± 0.2 when applying a cspr of 0.06 l/(cell × day) with fresh cd-u3 medium (vazquez-ramirez et al. 2018 ). different to the hipcop strategy fig. 2 progression of the mva-cr19 virus production for two hybrid fb/perfusion variants, hybrid 1 (a) and hybrid 2 (b). virus titers of whole cell lysates (filled circles) and permeate (crosses) are indicated for both variants. virus titers in the supernatant (triangles) were determined only for hybrid 2 (b). arrows: time points of bolus feeding proposed by hiller et al. (2017) , which operates at glucose limitation and a lactate consumption regime in cho cell cultivations, this strategy allowed for a perfusion control without reaching low glucose concentrations that might negatively affect the growth of cr.pix cells. after the cr.pix cells were cultivated to 27 × 10 6 cells/ ml, the v w was reduced from 0.6 to 0.3 l and 1 reactor volume was exchanged with fresh medium before virus infection (fig. 3a) . a final concentration of 47 × 10 6 cells/ml was measured just before infection (fig. 3b) . influenza a viruses are reported to replicate rapidly in cr.pix cells (jordan et al. 2016; lohr et al. 2009 ). an increase in ha titers is typically observed at about 6 hpi and, depending on the virus strain, maximum titers are obtained 24-36 hpi (lohr et al. 2009 ). based on this information, and in contrast to the production of mva virus, the fb phase was shortened (0-12 hpi), followed by a perfusion phase at a rate of one reactor volume per day (fig. 3a) . a process operated completely in perfusion and with total virus retention, as proposed by genzel et al. (2014) , was performed as a reference process (online resource 1). the hybrid cultivation yielded a maximum ha titer of 3.3 log hau/100 μl (3.8 × 10 10 virions/ml) at 60 hpi (fig. 4) . this represented a fivefold increase compared to the reference perfusion cultivation (fig. 4) . compared to a conventional batch process (table 2) , the increase was sevenfold. the implementation of the hybrid strategy in benchtop bioreactors showed a similar performance compared to analogous experiments in shake flasks and led to a clear improvement of virus yields towards perfusion (for hcd) and batch (for conventional cell densities). next, broader benefits in relation to reported production strategies with different cell substrates and its advantages for a potential industrial application were analyzed. although cd-u3 medium was not developed for perfusion processes (jordan et al. 2011) , medium consumption to achieve concentrations of about 50 × 10 6 cells/ml with cspr-based perfusion was moderate (6.15 reactor volumes for hybrid 1 and 1.98 reactor volumes for hybrid 2). despite the low medium utilization, the average cell-specific growth rates for both hybrid 1 and 2 cultivations were comparable to the 0.023 1/h achieved previously in shake flasks (vazquez-ramirez et al. 2018) . accordingly, it also took only about 8 days to reach a minimum vcd of 50 × 10 6 cells/ml. petiot et al. (2011) reported a medium utilization of about 3.5 reactor volumes to expand hek293 cells within 9 days from 0.25 to 15 × 10 6 cells/ml (before infection with influenza virus). genzel et al. (2014) reported a medium consumption of 11.3 reactor volumes to propagate age1.cr cells to 50 × 10 6 cells/ml before infection with influenza virus. therefore, the results here represent a significant reduction in medium consumption before virus infection, which is an important contribution towards lower cogs in large-scale production. both hybrid 1 and hybrid 2 variants simplify the production process because a single bioreactor can be used for cell expansion and virus propagation. in hybrid 1 cultivation, one-half of the cell suspension (0.3 l) was removed before infection at 50 × 10 6 cells/ml (fig. 1 a) .this cell suspension could possibly be used to start a second bioreactor in parallel. in contrast, in the hybrid 2 cultivation, cells were cultivated to 25 × 10 6 cells/ml in 0.6 l and concentrated to 50 × 10 6 cells/ml prior to infection (fig. 1 d) . in both cases, the subsequent fb phase required the addition of almost three times the starting volume to avoid substrate limitations. this ratio was lower than the 1:4 reported by pohlscheidt et al. (2008) for the high-yield production of parapoxvirus ovis at large scale, which-in addition-required transferring the cell suspension to a second larger bioreactor to perform the dilution steps. since the initial fb phase of the hybrid strategy seems to be a critical operation also for mva-cr19 virus propagation (vazquez-ramirez et al. 2018) , further studies could focus on the development of an optimized feed medium to enable a higher starting volume (preferably 60% of the maximum working volume) and a lower maximum dilution ratio (about 2:3) to simplify the hybrid strategy for implementation in large-scale bioreactors. overall, the established hybrid strategies for mva-cr19 virus production (table 2 , hybrid 1 and hybrid 2) resulted in a 10 to 100-fold increase in virus titers compared to the current standard production platform in cef cells (gilbert et al. 2005; meiser et al. 2003) . with respect to cultivations performed at conventional cell densities using cr.pix cells lohr et al. 2009; lohr 2014) , eb14 cells (guehenneux and pain 2005) , and eb66 cells (léon et al. 2016 ), up to tenfold higher titers were obtained. cell-specific virus yields obtained with the hybrid strategies (410 and 352 iu/cell) were also competitive regarding the 500 iu/cell obtained with cef cells (carroll and moss 1997) , the 50-200 iu/cell with cr.pix cells (lohr 2014) , and the 25-50 iu/cell with eb66 cells (léon et al. 2016 ) at conventional lower cell densities. batch production of mva virus with cr.pix cells lohr 2014) and eb66 cells (léon et al. 2016 ) requires more or less the same time and the same media volumes. accordingly, its volumetric productivity of about 2.0 × 10 10 iu/(l × day) is clearly surpassed by the 1.3 and 2.8 × 10 11 iu/(l × day) obtained with the hybrid strategy. applying such a strategy would allow for 100,000 doses per liter of cell-free supernatant, considering that single doses of 1 × 10 8 pfu (1 pfu = 1 iu) per individual are currently used in clinical studies involving recombinant mvabased vaccines (gomez et al. 2013) . due to its application as a viral vector, maintaining the infectious activity of mva is a critical quality attribute. it is promising that titers were found to remain stable from 72 to 120 hpi (fig. 2b ). this suggests a low virus inactivation rate for the specific cultivation conditions chosen. while continuous virus harvesting failed for the chosen atf system, the use of other cell retention devices including acoustic filter and settlers might be evaluated again for large-scale production to avoid product losses. one major property of the mva-cr19 virus, its capacity to propagate in true single-cell suspension cultures, may additionally help to facilitate the recovery of infectious units directly from the culture supernatant without the need of cell disruption (jordan et al. 2013) . the hybrid 2 cultivation showed that the maximum titer at 72 hpi accounted entirely for virus in the supernatant (fig. 2b) with most of the cells showing a viability > 70%. hence, a clarification step at this point with a carefully chosen cell retention system would suffice to recover the mva-cr19 virus from the bioreactor. based on the very high performance of the hybrid strategy in upstream processing and further options to reduce costs in downstream processing, it can be assumed that higher costs related to the implementation of bcomplex^perfusion processes (purchase of dedicated equipment and training of staff) can be more than compensated even at industrial scale. the perfusion control applied during the cell growth phase in the hybrid cultivation led to a μ max = 0.025 1/h and an overall μ mean = 0.019, which were in accordance with previous hcd bioreactor cultivations (table 1) . similar to the hybrid 2 process for mva-cr19 virus, on-line vcv estimations correlated well with off-line measurements up to late stages of the influenza a virus propagation phase (fig. 3b) . no glucose limitation nor significant lactate accumulation was observed for the ph-based perfusion control during the cell growth phase (fig. 3c) . the high average cspr of 0.119 nl/(cell × day) (table 1, fig. 3a ) obtained in the cell growth phase led to an increase in medium consumption (16.7 reactor volumes) compared to the reference perfusion process (3.9 reactor volumes). since the perfusion rates depended on the ph control of the cultivation, reducing the ph set point could further minimize the high medium exchange. despite the very high medium consumption, the hybrid strategy provided a cell-specific yield of 1300 virions/cell and a volumetric productivity of 5.4 × 10 11 virions/(l × day) ( table 2 ). this observation confirms that the cell density effect can be circumvented by a hybrid strategy for influenza a virus-infected cultures. the difference of 17% in volumetric productivity, with respect to the batch cultivation, can be explained by the rather high medium consumption during the cell growth phase of the hybrid cultivation. compared to the perfusion-only strategy, the hybrid fb/perfusion conferred a clear improvement in both cell-specific and volumetric productivity (table 2) , and showed comparable yields for influenza a virus with respect to the parental suspension cell line age1.cr (genzel et al. 2014) . process intensification by consequent use of hcd strategies in viral vaccine manufacturing can contribute to a stable supply of vaccines. in case conventional batch production processes are already established, the implementation of hcd strategies can significantly increase manufacturing capacities, e.g., in emerging and developing countries where vaccines are most urgently needed. a possible solution using a hybrid fb/ perfusion strategy during the virus production phase for mva and influenza a virus in small stirred tank bioreactors is described here. the application of this strategy resulted in a 7to 20-fold increase in virus titers without compromising cellspecific yields and volumetric productivities that often hinder the establishment of intensified processes. the high titers of 10 10 iu/ml obtained for mva virus demonstrated, in particular, the potential of this approach as an alternative to the current technology that relies on primary chicken embryo fibroblasts as a substrate. the results achieved here for the two different viruses may be also instructive for modernization of conventional approaches in viral vaccine production. modified vaccinia virus ankara (mva) as production platform for vaccines against influenza and other viral respiratory diseases viral vaccine production in cell culture therapeutic vaccination to treat chronic infectious diseases: current clinical developments using mva-based vaccines the avian eb66® cell line, application to vaccines, and therapeutic protein production the development and manufacture of influenza vaccines host range and cytopathogenicity of the highly attenuated mva strain of vaccinia virus: propagation and 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and next generation influenza vaccines: formulation and production strategies mva vaccination against smallpox -clinical tests with an attenuated live vaccinia virus strain (mva) bioreactors for high cell density and continuous multi-stage cultivations: options for process intensification in cell culture-based viral vaccine production highcell-density cultivations to increase mva virus production enhanced t cell-mediated protection against malaria in human challenges by using the recombinant poxviruses fp9 and modified vaccinia virus ankara influenza b virus reverse genetic backbones with improved growth properties in the eb66® cell line as basis for vaccine seed virus generation perfusion seed cultures improve biopharmaceutical fedbatch production capacity and product quality concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity acknowledgements this project is financially supported by the mexican national council of science and technology (conacyt) and the german academic exchange service (daad).funding information open access funding provided by max planck society. conflict of interest this article does not contain any studies with human participants or animals performed by any of the authors.the authors declare that they have no conflicts of interest.open access this article is distributed under the terms of the creative comm ons attribution 4.0 international license (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-287602-vda01gj6 authors: jin, yu-bei; yang, wen-tao; shi, chun-wei; feng, bo; huang, ke-yan; zhao, guang-xun; li, qiong-yan; xie, jing; huang, hai-bin; jiang, yan-long; wang, jian-zhong; wang, guan; kang, yuan-huan; yang, gui-lian; wang, chun-feng title: immune responses induced by recombinant lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets date: 2018-07-18 journal: appl microbiol biotechnol doi: 10.1007/s00253-018-9205-0 sha: doc_id: 287602 cord_uid: vda01gj6 transmissible gastroenteritis coronavirus (tgev) is one of the most severe threats to the swine industry. in this study, we constructed a suite of recombinant lactobacillus plantarum with surface displaying the spike (s) protein coming from tgev and fused with dc cells targeting peptides (dcpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. our research results found that the recombinant lactobacillus plantarum (nc8-psip409-pgsa-s-dcpep) group expressing s fused with dcpep could not only significantly increase the percentages of mhc-ii(+)cd80(+) b cells and cd3(+)cd4(+) t cells but also the number of iga(+) b cells and cd3(+)cd4(+) t cells of ileum lamina propria, which elevated the specific secretory immunoglobulin a (siga) titers in feces and igg titers in serum. taken together, these results suggest that nc8-psip409-pgsa-s-dcpep expressing the s of tgev fused with dcpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of tgev vaccines. transmissible gastroenteritis of pigs is a highly contagious disease caused by porcine transmissible gastroenteritis virus and has clinical symptoms of severe diarrhea, vomiting, dehydration, and high death rate for newborn piglets (peng et al. 2017) . in 1946, doyle and hutchings for the first time reported tge in the united states (doyle and hutchings 1946) , and it has now become a worldwide swine disease and has caused great harm to the global pig industry . tgev is one of the main causes of piglet disease and death today. the spike (s) protein of transmissible gastroenteritis virus encoded by the s gene of tgev is located in the outermost organelles and the main antigen protein of tgev; it carries the major b lymphocyte epitopes and is the only structural protein that induces the body to produce neutralizing antibodies and provide immunoprotection (krimmling et al. 2017) . tgev is also a cell-dependent viral antigen, and t cells can respond to whole virus particles and cause a cellular immune response (chen et al. 2016) . lactobacillus is a group of bacteria that can ferment carbohydrates, producing large amounts of lactic acid (könig and fröhlich 2017) . they are long-term colonizers of the human or animal gut and have many benefits to the body, and they are recognized internationally as food-grade safety microbes (saad et al. 2013) . lactobacillus has become the best choice for the expression of heterologous proteins and live vector vaccines presenting antigens in the field of genetically engineered vaccines because of its many advantages, such as easy culture, simple operation, and high safety (trombert 2015) . the lactobacillus plantarum nc8 strain was a strain of lactobacillus that was isolated from grass silage in the 1980s (axelsson et al. 2012) . now, it is used as a model strain in the development of genetic tools, for instance, transformation, conjugation, and expression vectors (yang et al. 2017a) . compared with lactobacillus of animal origin, such as lactococcus lactis, due to its resistance to stress and suitability as a host bacterium for expressing foreign protein, lactobacillus plantarum has gained increasing attention (jiang et al. 2015) . dendritic cell peptides (dcpep) are derived from phage oligopeptides, which can significantly enhance the immune response (yang et al. 2017b ). dcpep has been considered to be a bridge between adaptive and host immunity because of its potent antigen-presenting ability and important role in inactivating and modulating the immune response (mohamadzadeh et al. 2008) . it can effectively capture foreign antigens and autoantigens and then present them to t cells, which induce different immune responses according to the environment . poly-γ-glutamic acetase synthase a (pgsa) is a constituent protein of the polyglutamate synthetase (pga) system of bacillus subtilis that can act as a bacterial surface display component and stably anchor-related enzyme systems to the surface of the cell membrane (narita et al. 2006) . given the characteristics of the pgsa protein, it has been applied to a variety of prokaryotic protein surface displays. in particular, it has been successfully applied in gram-positive receptor strains such as lactic acid bacteria, which provide a theoretical basis for us to study how to anchor exogenous proteins on the cell wall surface of lactobacillus plantarum (lei et al. 2015; yang et al. 2017c) . previously, we successfully constructed recombinant l. plantarum nc8 (nc8-psip409-pgsa-s-dcpep) to enable the s antigen to be effectively recognized by the dc in the intestinal mucosa and to be exhibited on the surface of l. plantarum, and recombinant nc8-psip409-pgsa-s-dcpep can successfully express pgsa-s-dcpep displayed on the cell wall surface of l. plantarum (data not published). in the present study, oral administration of nc8-psip409-pgsa-s-dcpep significantly increased the secretion of il-4, ifn-γ, secretory immunoglobulin a (siga), and igg and the number of mhc-ii + cd80 + b cells and cd3 + cd4 + t cells in piglets, which provided a potential to protect against tgev challenge. this provides a strategy for the preparation of oral lactic acid bacteria vaccines to prevent transmissible gastroenteritis of pigs. the recombinant l. plantarum (for the delivery of s protein) used in this study was previously constructed by our laboratory. all the recombinant l. plantarum were cultured in man rogosa sharpe (mrs) medium at 30°c without shaking. when required, erythromycin was added to l. plantarum strain nc8 (ccug 61730) at 10 μg/ml. a total of 25 one-month-old tgev-seronegative crossbreed junmu1 white pigs were provided by jilin university pig farm and assigned to five groups (n = 5) named saline, nc8-psip409-pgsa, nc8-psip409-pgsa-s-ctrlpep (the s gene fragment (genbank accession no. kt696544, source-20,365 to 22,410)/spike protein genbank accession no. amb66488, source-1 to 682 and the control peptide (epihpettftnn)), nc8-psip409-pgsa-s-dcpep (dc peptide (fypsyhstpqrp)) (shao-hua et al. 2016) , and tgev inactivated vaccine. groups were housed in a specific pathogen-free (spf) environment. the piglets of the same group were placed in a uniform environment; however, they were separated into rooms and fed a balanced diet with free access to water. the piglets were administered orally with 5 ml saline, 10 10 cfu nc8-psip409-pgsa, 10 10 cfu nc8-psip409-pgsa-s-ctrlpep, 10 10 cfu nc8-psip409-pgsa-s-dcpep, and 2 ml inactivated tgev, respectively, approximately twice at 7-day intervals. the feces and serum were collected every week after the initial immunity. the blood samples were incubated at 4°c overnight without shaking and then centrifuged at 4°c and 3000 rpm for 10 min, and the serum was obtained and stored at 80°c for subsequent analysis. the feces were collected as previously published . the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. all group piglets were euthanized at 21 days after secondary immunization, and the intestinal segments were collected as previously described but with minor alterations (zhao et al. 2014) . in detail, upon washing with ice-cold pbs, the fixed tissues were immediately embedded in oct (embedding medium for frozen tissue specimens; sakura, usa) and then stored at − 80°c until further use. frozen tissue sections (5 μm thick) were cut on a frozen slicer (leica cm 3050s cryostat, germany) and transferred to poly-l-lysine-coated microscope slides. the slides were air-dried and stored for up to 4 weeks at − 80°c before immunofluorescence staining. the animal management procedures and all laboratory procedures abided by the demands of the animal care and ethics committees of jilin agriculture university, china. the levels of tgev-specific siga in feces and igg in the sera were identified by enzyme-linked immunosorbent assay (elisa) according to previously published methods mou et al. 2015) . moreover, the levels of il-4 and il-17 in serum were determined by elisa according to the manufacturer's instructions (lvye biotechnology, china) with minor alterations. the difference between the original manufacturer's recommendations and the changed was only in the sample processing method. the changed was only that all samples were added at twofold dilutions from 1/2 to 1/2048 in diluted buffer, and the other steps followed the instructions in the kits. finally, the absorbance at 450 nm was read with a microplate reader (biotek, usa). single-cell suspensions were prepared from spleens, mesenteric lymph nodes (mlns), peyer's patches (pps), and ileum lamina propria (ilp) using modifications of previously published methods (rios et al. 2016) , and then stained with specific antibodies as described previously (sinkora and sinkorova 2014) . the percentages of cd3 + cd4 + t cells and mhc-ii + cd80 + igm + b cells were evaluated by facs (bd lsrfortessa, usa). mouse antipig mabs were used as primary immunoreagents. singlecell suspensions from spleens and pps were stained with specific antibodies for anti-igm (m160, igg1), anti-mhc-ii (msa3, igg2a), and anti-cd80 (mem-233, igg1) to analyze the percentages of mhc-ii + cd80 + igm + b cells. goat polyclonal abs specific for mouse ig subclasses labeled with allophycocyanin (apc), allophycocyanin/ cyanine 7 tandem complex (apc-cy7), and peridininchlorophyll-protein complex (percp) were used as secondary antibodies. all fluorescent secondary antibodies were purchased from southern biotech (usa). the single-cell suspensions from mlns were stained with anti-cd3 (fitc) (bb23-8e6-8c8; bd pharmingen), anti-cd4 (apc) (74-12-4; bd pharmingen), and anti-cd8(pe) (76-2-11; bd pharmingen) to analyze the percentages of cd3 + cd4 + t cells. mlns are mainly used to analyze the immunoprotection supplied by lactic acid bacteria vaccines in mucosal immunity. to examine the viability of t cells from the mlns, t-cell proliferation was performed. single-lymphocyte suspensions of each group of piglets (n = 5) were prepared from the mlns when the piglets were euthanized as previously published (jiang et al. 2014) with slight changes. in detail, the lymphocytes were incubated in triplicate in 96-well plates at 5 × 10 5 cells/well in roswell park memorial institute 1640 medium (rpmi-1640) containing 10% fetal calf serum (fcs) and stimulated with 5 μg/ml phytohemagglutinin (pha) and culture medium as a negative control in a 5% co 2 incubator (thermoscientific, usa) at 37°c for 3 days. a thiazolyl blue (mts) solution was added to each well, and 10 μl was added to each well to develop the color after 3 days. the od490 values were then detected using a microplate reader (biotek, usa), and the value of the negative control wells was set to zero after 4 h of incubation. the lymphocytes from the spleens, mlns, pps, and ilp were determined by real-time rt-pcr (qpcr) using a cfx96tm real-time pcr detection system (bio-rad, usa). qpcr was used to quantify the messenger rna (mrna) of cd80, cd86, cd40, tlr-2, tlr-9, il-4, il-17, ifn-γ, tgf-β, b-cell activating factor (baff), and a proliferationinducing ligand (april) in the total rna isolated from 1 × 10 6 lymphocytes of spleens, mlns, pps, and ileum lamina propria using an rna extraction kit according to the manufacturer's recommendations (takara, japan). total rna concentration was identified with biotek epoch 2 microplate reader (biotek, usa). the total rna were reverse transcribed using the primescript™ rt reagent kit with gdna eraser (takara, japan) according to the manufacturer's instructions. the specific primer sequences are listed in table 1 . in the end, the 2 − δδct method was utilized to calculate relative gene expression compared with the β-actin gene control (livak and schmittgen 2001) . the cryosections were incubated with anti-pig cd3, antipig cd8, anti-pig cd4 (three-antibody combination; bd biosciences, usa), or anti-pig iga (fitc) (abcam, uk) as described previously (subramaniam et al. 2017 ) but with some changes. briefly, phosphate-buffered saline (pbs) containing 10% pig serum was used to block fc receptors for 30 min. combinations of cd3, cd4, cd8, or iga mabs were added to the slides overnight at 4°c; subsequently, the slides were washed three times for 5 min each time, with fresh changes of tbs-tween. the cell nuclei were then stained with 4,6-diamidino-2-phenylindole (dapi) solution (invitrogen, usa) for 5 min and washed three times for 5 min each time, with fresh changes of tbs-tween. the slides were imaged by confocal microscopy (zeiss lsm710, germany), and the imaging results were analyzed using zeiss 2012 (blue edition). if not additionally declared, the data were presented as arithmetic mean values ± sem, and statistical analysis was performed by one-way analyses of variance (anovas) using graphpad prism 5.0 software. p < 0.05 was considered statistically significant. the rna of lymphocytes from spleens (sl), mlns (ml), pps (pl), and ilp (il) were extracted to analyze the expression of tlr-2 and tlr-9 by real-time rt-pcr (fig. 1) . the tlr-2 and tlr-9 expression in ilp, pps, and mlns was higher in the nc8-psip409-pgsa-s-dcpep group than in the other groups compared with the saline, nc8-psip409-pgsa, nc8-psip409-pgsa-s-ctrlpep, and tgev inactivated vaccines (fig. 2) . moreover, the results also showed that tlr-2 and tlr-9 of lymphocytes from spleens displayed the same trend in the nc8-psip409-pgsa-s-dcpep group than in the other groups (fig. 2) . therefore, the results suggested that nc8-psip409-pgsa-s-dcpep could enhance innate immune responses in piglets. (1 × 10 10 cfu in 2 ml), saline (2 ml), or tgev inactivated vaccine (2 ml), respectively, for each piglet on days 1-3 and 6-8 the rna of lymphocytes from ilp, pps, mlns, and spleens were extracted to analyze the expression of these costimulatory molecules cd40, cd80, and cd86 in b cells by real-time rt-pcr. furthermore, to assess the expression of cd80 and mhc-ii on the surface of b cells, we performed flow cytometry. all piglets were euthanized 21 days after secondary immunization. the results showed that nc8-psip409-pgsa-s-dcpep significantly improved the expression of cd40, cd80, and cd86 in the b cells from ilp, pps, mlns, and spleens by real-time rt-pcr compared with the other groups ( fig. 3a-c) . in addition, the results found that nc8-psip409-pgsa-s-dcpep could also significantly increase the expression of cd80 and mhc-ii on the surface of b cells from pps compared with the groups of saline (p < 0.001), nc8-psip409-pgsa (p < 0.001), nc8-psip409-pgsa-s-ctrlpep (p < 0.001) (fig. 3d) , and spleens compared with the groups of saline (p < 0.001), nc8-psip409-pgsa (p < 0.05), and nc8-psip409-pgsa-s-ctrlpep (p < 0.05) by flow cytometry (fig. 3e) ; however, nc8-psip409-pgsa-s-dcpep was not significant compared with the group of tgev inactivated vaccine about increasing the expression of cd80 and mhc-ii on the surface of b cells from pps and spleens (fig. 3d, e) . in summary, the all data suggested that nc8-psip409-pgsa-s-dcpep could induce b-cell activation not only mucosally but also systemically in piglets. the number of iga + b cells in ilp coming from all groups of piglets was determined by immunofluorescence. the results showed that nc8-psip409-pgsa-s-dcpep could significantly increase the number of iga + b cells in ilp compared with the groups of saline (p < 0.001), nc8-psip409-pgsa (p < 0.001), nc8-psip409-pgsa-s-ctrlpep (p < 0.01), and tgev inactivated vaccine (p < 0.05) (fig. 4 ). in addition, the titers of anti-tgev siga in feces and anti-tgev igg titers in the serum of piglets after oral immunization were determined by elisa. we found that the tgev-specific siga antibody titers in feces induced by nc8-psip409-pgsa-s-dcpep significantly increased compared to saline (p < 0.001) and nc8-psip409-pgsa (p < 0.001) from 7 days to 21 days and that the value reached the maximum at 14 days after initial immunity (fig. 5a ). in addition, nc8-psip409-pgsa-s-dcpep did not significantly increase the tgev-specific siga antibody titers in feces compared to tgev inactivated vaccine (positive control) (p > 0.05) (fig. 5a) . the results also showed that nc8-psip409-pgsa-s-dcpep could also significantly induce specific igg antibody titers in serum compared with others ( fig. 5b) . however, there were no significant differences between nc8-psip409-pgsa-s-dcpep and tgev inactivated vaccine (p > 0.05) (fig. 5b) . therefore, the data suggested that nc8-psip409-pgsa-s-dcpep served as the vaccine to immunize the piglets and could induce local mucosal immune responses and systemic immune responses. in addition, to further demonstrate that the recombinant l. plantarum could influence cell-mediated immunity, we prepared a single-cell suspension of mlns lymphocytes coming from piglets immunized with saline, recombinant l. plantarum, and tgev inactivated vaccine groups 28 days after the boost immunization and then carried out a cell proliferation test. the results showed that nc8-psip409-pgsa-s-dcpep resulted in significantly higher levels of t-cell proliferative responses than saline (p < 0.05) (fig. 6) . however, there were no significant the mean values ± sem of three independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. the error bars represent standard deviations differences compared with the nc8-psip409-pgsa, nc8-psip409-pgsa-s-ctrlpep, and tgev inactivated vaccine (fig. 6 ). this result implied that nc8-psip409-pgsa-s-dcpep could enhance the viability of t cells of mlns in piglets. the single-cell suspensions from the mlns were stained with anti-cd3 antibody, anti-cd4 antibody and anti-cd8 antibody to detect by facs. as shown in fig. 7 , nc8-psip409-pgsa-s-dcpep significantly increased the percentages of cd3 + cd4 + t cells in mlns compared to the groups of saline (p < 0.001), nc8-psip409-pgsa (p < 0.001), nc8-psip409-pgsa-s-ctrlpep (p < 0.01), and tgev inactivated vaccine (p < 0.05). moreover, the number of cd3 + cd4 + t cells in the ilp coming from all groups of piglets was determined by immunofluorescence. the results showed that nc8-psip409-pgsa-s-dcpep could significantly increase the number of cd3 + cd4 + t cells in ilp (fig. 8) . these results indicated that oral immunization with nc8-psip409-pgsa-s-dcpep could effectively increase ileum local lymphocyte cells and induce local mucosal immune responses in piglets. the cytokine expression was also detected in the splenic lymphocytes (sl), mesenteric lymph node lymphocytes (ml), and ileum lamina propria lymphocytes (il) by qpcr or elisa in all piglets. in this study, the expressions of il-4 and il-17 in serum induced by nc8-psip409-pgsa-s-dcpep were higher than saline (p < 0.01), nc8-psip409-pgsa (p < 0.01), and nc8-psip409-pgsa-s-ctrlpep (p < 0.05) by elisa (fig. 9a, b) . in addition, the il-4, il-17, ifn-γ, and tgf-β expressions of sl, ml, and il induced by nc8-psip409-pgsa-s-dcpep were higher than those induced by the saline, nc8-psip409-pgsa, and nc8-psip409-pgsa-s-ctrlpep by qpcr (fig. 9c-f) ; however, nc8psip409-pgsa-s-dcpep was not significantly different compared with the nc8-psip409-pgsa and nc8-psip409-pgsa-s-ctrlpep on the il-17 expression in il (fig. 9d) . besides, we also found that the expressions of baff and april in sl, ml, and il were the higher in the nc8-psip409-pgsa-s-dcpep group than in the other groups compared with the saline, nc8-psip409-pgsa, and nc8-psip409-pgsa-s-ctrlpep by qpcr (fig. 9g, h) . moreover, the baff and april expressions in the ml and il in the nc8-psip409-pgsa-s-dcpep group were significantly enhanced compared to the tgev inactivated vaccine; however, there was no significant difference in sl (fig. 9g, h) . the higher expression of baff and april suggested that nc8-psip409-pgsa-s-dcpep could effectively promote b-cell maturation and activation. lactobacillus plantarum is a well-characterized bacterium that is a particularly popular probiotic used to express heterogeneous proteins and deliver targeted antigens . in a previous research report, due to the use of the intracellular expression vector psip409 to express target antigens aiming to protect against newcastle disease virus (ndv), the research results did not achieve the expected results (jiang et al. 2015) . to overcome these hurdles, as a powerful technology, surface display may be a good choice to express heterogeneous peptides and proteins on cells, making use of natural functional components of microorganisms (raha et al. 2005; kuczkowska et al. 2016 ). poly-γ-glutamic acid synthetase a (pgsa), which is a constituent protein of the polyglutamate synthetase system (pga) of bacillus subtilis, has been widely used to anchor foreigner protective antigens on the surface of lactic acid bacteria (sewaki 2010; lei et al. 2015) . in previous studies, we successfully used surface display technology to express a number of targeted proteins, such as hemagglutinin subunit 2 (ha2) of avian influenza virus (aiv), and murine il-10 and spike protein (s) of porcine epidemic diarrhea virus (pedv) (cai et al. 2016; huang et al. 2017; jiang et al. 2017 ). because dc targeting strategies could stimulate stronger and lasting immune responses, they have attracted the eyes of an increasing number of people. in the present study, to reduce the number of vaccinations of l. plantarum, which served as oral administration, the specific 12-mer dc-binding peptides were utilized to target dcs from the bacteriophage library. previous studies have found that anthrax pa or hepatitis c virus (hcv) ns3, which genetically fused dc-binding peptides, could efficiently deliver antigens to dcs (shao-hua et al. 2016) . in previous reports, this vaccine strategy offered a variety of benefits in that the constructed l. plantarum expression of protein-fused dcpep could activate mucosal dcs, b cells, and t cells to induce the immunological response and could regulate inflammatory responses that took place in a mucosal microenvironment (steinman and idoyaga 2010) . our laboratory has previously studied yang et al. 2016 ) the effects of recombinant lactic acid bacteria expressing target antigens and dcpep on mhc-ii + cd80 + dcs; however, the impact of recombinant lactic acid bacteria cells has not been studied in mhc-ii + cd80 + b (such cells not only can secrete siga involved in mucosal immunity but also can act as antigen-presenting cells) (adler et al. 2017; mizoguchi and bhan 2017) . therefore, in this study, we mainly researched the effects of recombinant lactic acid bacteria nc8-psip409-pgsa-s-dcpep expressing dcpep and target antigen s protein on mhc-ii + cd80 + b cells, cd3 + cd4 + cells related to mucosal immune responses, and anti-tgevspecific antibodies. in this study, the results showed that the recombinant lactic acid bacteria nc8-psip409-pgsa-s-dcpep could raise not only the rate of mhc-ii + cd80 + b cells in pps and spleens by flow cytometry but also the number iga + b cells in ileum lamina propria by immunofluorescence and t cells in the mesentery by flow cytometry (figs. 3, 4, and 7) . this research also showed that nc8-psip409-pgsa-s-dcpep could increase s-specific siga antibody titers in fecal matter to produce mucosal immune responses and s-specific igg antibody titers in serum to produce humoral immune responses by elisa analysis (fig. 5) . the researchers found that immunization with antigenfused dc targeting peptides could notably induce cd3 + cd4 + t cells to expand and proliferate (lahoud et al. ; similarly, our results showed that nc8-psip409-pgsa-s-dcpep expressing s-dcpep also significantly boosted the number of cd3 + cd4 + t cells in mlns compared with s alone (fig. 7) . it has been considered that cd3 + cd4 + t cells serving as a type of t-helper cells could promote other immune cells to mature and activate by secreting a variety of cytokines. the primary liability of the significant production of mucosal siga in the nc8-psip409-pgsa-s-dcpep group should be borne by observed t-cell expansion. this may be the cause for recombinant lactic acid bacteria nc8-psip409-pgsa-s-dcpep expressing dcpep and target antigens s protein being able to better assist recombinant antigen-induced dendritic cell proliferation and activation in piglets in vivo. dcs captured the dcpep binding targeted antigen s protein and then presented the antigens to t cells; by secreting cytokines, th cells activated the b cells . another reasonable explanation is that dcpep may also be recognized by b cells which some b cells also have antigenpresenting function, which subsequently presented the antigens to t cells with apc function. of course, the specific mechanism needs to be further studied. ifn-γ, which is a cytokine, could enhance phagocytic activity to efficiently kill pathogens and is produced by nk cells and t lymphocytes. it was reported that ifn-γ could cause th1 responses to protect against pathogen infection by adjusting chemotaxis and enhancing antigen presentation (schroder et al. 2004 ). il-4 promotes the expression of mhc-ii and cd40 in b cells and enhances the ability of b cells to present antigen so that the immune system can produce an immune response to antigen stimulation. the th1 and th2 cell responses are related to the secretion of ifn-γ and il-4, respectively. as a consequence, in this study, the secretion levels of ifn-γ and il-4 were analyzed in the immunized animals to indirectly reflect the capacity of the vaccine to induce the th1 or th2 response. in addition, the balance between th1 and th2 responses was evaluated by the secretion levels of these cytokines (chen et al. 2010) . in this study, compared to the saline control group in the piglets, we found that nc8-psip409-pgsa-s-dcpep could significantly induce the secretion of ifn-γ and il-4, indicating that nc8-psip409-pgsa-s-dcpep significantly heightened the immunogenicity of the tgev vaccine and triggered the immune response of th1-and th2-type cells (fig. 9) . the results also suggested that nc8-psip409-pgsa-s-dcpep administered orally to piglets had a powerful potentiation influence on both humoral and cellular immunity. a previous study showed that oral administration of lactobacillus fermentum cect5716 notably heightened not only the production of th1-type cytokines in serum but also specific siga antibody responses to influenza (olivares et al. 2007 ). the increased percentages of il-4-secreting th2 cells then possibly stimulated the differentiation of iga + b cells and increased production of mucosal siga antibodies. in addition, the primary function of th2 cells is to induce b-cell proliferation and produce antibodies that are related to humoral immunity. it was reported that il-17 has significant functions that participate in pro-and anti-inflammatory effects . previous research showed that recombinant lactobacillus induced the expression of il-17 in both systemic and mucosal immune responses to protect against tgev infection (jiang et al. 2014) . similarly, in this study, we found that piglets immunized with nc8-psip409-pgsa-s-dcpep could remarkably induce the expression of il-17 in spleen cells (systematic immune responses) and mesenteric lymph node cells (mucosal immune responses) compared with other groups (fig. 9 ). regulatory t (treg) cells take part in the regulation of anti-inflammatory responses primarily through the secretion of cytokines such as il-10 and tgf-β (noack and miossec 2014) . previous studies have found that the oral administration of lactobacillus casei can increase the expression of tgf-β in blood, and this is essential for th17 differentiation in the spleen (jiang et al. 2014) . microbe-associated molecular patterns or soluble factors from probiotic bacterial genome dna, such as probiotic bacterial genome dna cpg, may regulate immunoregulatory effects and enhance iga. there was a report that compared with the vaccinated group, which was not colonized in piglets, the vaccinated probiotic colonized in piglets dramatically increased small intestinal tlr9 expression (vlasova et al. 2013) . tlr9 recognizes bacterial cpg motifs and the higher expression of tlr9 coming from mncs was very important for mucosal iga to participate in the immune response. a previous study showed that toll-like receptors (tlrs) play an important role in the activation of dcs using recombinant lactobacillus (kathania et al. 2013) . recombinant lactobacillus has been reported to serve as a vaccine to effectively inhibit the expression of tlr in piglets . however, in this study, we also evaluated the expression of tlr-2 and tlr-9 in piglets immunized with recombinant l. plantarum by real-time rt-pcr analysis and found that recombinant l. plantarum could increase the number of tlr-2 and tlr-9 expression in pigs to stimulate the host mucosal immune system (fig. 2) . it is likely that lactobacilli induced the expression of tlr-2 and tlr-9, which is related to the higher lactobacilli quantity (wen et al. 2009 ). it is well known that the expression levels of cytokines and tlr in splenic lymphocytes are indicators of systemic immunity, but the expression of cytokines and tlr in the cells coming from mlns is concerned with local and mucosal immune responses. baff and april are usually secreted by not only monocytes and intestinal epithelial cells but also t cells (fagarasan et al. 2010 ). baff has a strong b-cell chemotaxis and can induce activated b cells to secrete large amounts of igg, iga, and igm as costimulatory factors for b-cell proliferation and differentiation in vitro, which can help the immature b cells of peripheral blood survive and differentiate into mature b cells in vivo (boneparth and davidson 2012) . a previous study showed that piglets colonized by lactobacillus rhamnosus showed that probiotic treatment enhanced the expression of april in the gut but had no influence on the expression of baff in mncs compared with control groups (kandasamy et al. 2014) . in this study, we found that recombinant lactic acid bacteria nc8-psip409-pgsa-s-dcpep also boosted april and baff expression in the gut (fig. 9) . a previous study reported that the cd40-cd40l and cd28-cd80 pathways are essential for the activation of t cells and activation of polyclonal b cells (tokunaga et al. 2005) . this research also found that recombinant lactic acid bacteria nc8-psip409-pgsa-s-dcpep could improve the expression of cd40 and cd80/cd86 (fig. 3) . the above results provided a reason for why nc8-psip409-pgsa-s-dcpep could enhance the number of iga + b cells and cd3 + cd4 + t cells in the ilp. it is well known that the mucosal immune response is considered the first barrier function to neutralize viruses, including tgev. previous research studies have already demonstrated that siga participates in the response protecting against disease at mucosal surfaces (liu et al. 2011) . siga agglutinates and incapacitates pathogens, by which it inhibits pathogen adhesion to the mucosal surface and is easily cleared in the secretions. therefore, siga disenables pathogens to interact with epithelial cell receptors and inhibits the assembly of viral particles within the host cell cytoplasm (kurashima and kiyono 2017) . recombinant lactic acid bacteria can induce the production of siga in the intestines. in our research, we found that recombinant lactobacillus nc8-psip409-pgsa-s-dcpep could significantly increase the titer of anti-tgev siga in feces and anti-tgev igg titer in the serum of piglets after oral immunization, comparing the different versions of nc8-psip409-pgsa, nc8-psip409-pgsa-s-ctrlpep, and nc8-psip409-pgsa-s-dcpep (fig. 5 ). in addition, it is worth noting that the levels of specific antibodies induced by nc8-psip409-pgsa-s-dcpep could continue 28 days at a high level. this may be related to lactobacillus colonization in the intestinal tract. comparing the different versions of nc8-psip409-pgsa, nc8-psip409-pgsa-s-ctrlpep, nc8-psip409-pgsa-s-dcpep, and tgev inactivated vaccines, upon immunization, we also found that nc8-psip409-pgsa-s-dcpep triggered expected immune responses between b and t cells, and as expected, the expression of siga was higher and continual. in addition, it has been shown that lymphocyte proliferation occurs in the ileum lamina propria of piglets immunized with an oral dose of recombinant l. plantarum. it was further suggested that recombinant lactobacillus could induce mucosal immune responses in piglets. in summary, all results recommended that the nc8-psip409-pgsa-s-dcpep expressing the s of tgev fused with dcpep could effectively induce immune responses, including mucosal immune and systemic immune responses. in conclusion, this study suggested that immunized piglets with nc8-psip409-pgsa-s-dcpep could enhance the percentages of mhc-ii + cd80 + b cells and cd3 + cd4 + t cells and induce the expression of cytokines to initiate immune responses. furthermore, nc8-psip409-pgsa-s-dcpep could significantly raise not only the specific siga titers in feces but also igg titers in serum. the nc8-psip409-pgsa-s-dcpep provided a feasible original strategy and approach for the design of tgev vaccines. fig. 9 analysis of the expression of cytokines. the levels of cytokines il-4 (a) and ifn-γ (b) in serum were detected by respective elisa kits. in addition, the rna of lymphocytes from spleens (sl), mlns (ml), and ilp (il) were extracted to analyze the expression of il-4 (c), il-17 (d), ifn-γ (e), tgf-β (f), baff (g), and april (h) by real-time rt-pcr. the mean values ± sem of three independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. the error bars represent standard deviations conflicts of interest the authors declare that there are no competing interests. ethical approval all applicable international and national guidelines for the care and use of piglets were followed. the other function: class ii-restricted antigen presentation by b cells genome sequence of the naturally plasmid-free lactobacillus plantarum strain nc8 (ccug 61730) b-cell activating factor targeted therapy and lupus surface-displayed il-10 by recombinant lactobacillus plantarum reduces th1 responses of raw264.7 cells stimulated with poly(i:c) or lps baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of cd8+ t-cell migration to the porcine gut a transmissible 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cells by langerin-expressing dendritic cells targeted with porcine epidemic diarrhea virus spike protein domains in pigs down-regulation of cd40 and cd80 on b cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab recombinant lactic acid bacteria as delivery vectors of heterologous antigens: the future of vaccination? lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs porcine epidemic diarrhea in china toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs cross-protective efficacy of dendritic cells targeting conserved influenza virus antigen expressed by lactobacillus plantarum construction and immunological evaluation of recombinant lactobacillus plantarum expressing so7 of eimeria tenella fusion dc-targeting peptide protection of chickens against h9n2 avian influenza virus challenge with recombinant lactobacillus plantarum expressing conserved antigens recombinant lactobacillus plantarum expressing ha2 antigen elicits protective immunity against h9n2 avian influenza virus in chickens effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen key: cord-011012-5mev3otu authors: rathore, abhishek singh; sarker, animesh; gupta, rinkoo devi title: production and immunogenicity of fubc subunit protein redesigned from denv envelope protein date: 2020-03-30 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10541-y sha: doc_id: 11012 cord_uid: 5mev3otu dengue virus (denv) is a vector-borne human pathogen that usually causes dengue fever; however, sometime it leads to deadly complications such as dengue with warning signs (dws+) and severe dengue (sd). several studies have shown that fusion (fu) and bc loop of denv envelope domain ii are highly conserved and consist some of the most dominant antigenic epitopes. therefore, in this study, fu and bc loops were joined together to develop a short recombinant protein as an alternative of whole denv envelope protein, and its immunogenic potential as fusion peptide was estimated. for de novo designing of the antigen, fu and bc peptides were linked with an optimised linker so that the three dimensional conformation was maintained as it is in denv envelope protein. the redesigned fubc protein was expressed in e. coli and purified. subsequently, structural integrity of the purified protein was verified by cd spectroscopy. to characterise immune responses against recombinant fubc protein, balb/c mice were subcutaneously injected with emulsified antigen preparation. it was observed by elisa that fubc fusion protein elicited higher serum igg antibody response either in the presence or in absence of freund’s adjuvant in comparison to the immune response of fu and bc peptides separately. furthermore, the binding of fubc protein with mice antisera was validated by spr analysis. these results suggest that fu and bc epitope-based recombinant fusion protein could be a potential candidate towards the development of the effective subunit vaccine against denv. electronic supplementary material: the online version of this article (10.1007/s00253-020-10541-y) contains supplementary material, which is available to authorized users. in the recent decades, dengue has emerged as one of the world's most dominant tropical diseases (guzman et al. 2016) with around 390 million (95% credible interval 284 to 528 million) cases recorded per year, of which only 96 million (95% credible interval 67 to 136 million) cases were manifested clinically (giraldo-garcía and castaño-osorio, 2019; bhatt et al. 2013 ). according to world health organization (who), an estimate of 3.9 billion people in 128 countries are living in areas with a high risk of dengue infection (brady et al. 2012 ). the recently estimated annual 390 million dengue cases reveals that the dengue disease burden has tripled as compared to previous predictions of 50 to 100 million reported cases without dengue warning signs. nonetheless, 250,000 to 500,000 patients were hospitalised due to dengue with warning signs (dws+) and severe dengue (sd), and the total annual cost of dengue burden was estimated globally around us$ 8.9 billion (giraldo-garcía and castaño-osorio, 2019; guzman et al. 2016) . in spite of having paucity of effective vaccines and drugs to expel dengue comprehensively, still it's enduring a big challenge to human (martina et al. 2009 ). therefore, next-generation vaccine strategies such as inactivated purified virus, dna or protein-based subunit vaccines, and their fusion chimeras are now going under investigation and some of them even under clinical trials (coller et al. 2011; danko et al. 2011; liu et al. 2015) . recently, dengvaxia (cyd-tdv), a tetravalent live attenuated vaccine has been approved for use in some of the highly dengue endemic areas where the sero-prevalence is higher than 70% (guy et al. 2015; pang et al. 2018 ). according to abhishek singh rathore and animesh sarker contributed equally to this work. electronic supplementary material the online version of this article (https://doi.org/10.1007/s00253-020-10541-y) contains supplementary material, which is available to authorized users. who, it has shown significant efficacy and acceptable safety profile during clinical trials in seropositive individuals; however, it carries a risk of severe dengue infection in seronegative individuals, and also failed to confer subsequent protection in denv-2 positive people in areas with less exposure of dengue (arredondo-garcía et al. 2018; durbin et al. 2011; guy et al. 2011) . several other vaccine candidates were also formulated based on live-attenuated dengue viruses (e.g. tv003, tv005) and inactivated purified virus (e.g. dpiv), of which some have already completed clinical trials and some are currently going under phase ii to iii clinical trials (whitehead et al. 2017; diaz et al. 2018 ). on the other hand, dna or proteinbased alternative subunit vaccines (e.g. tvdv, den-80) and dengue-flavivirus chimeras (e.g. ediii-p64k, 80e-stf2, ediii-hbsag) are going either under preclinical study or phase i clinical test (govindarajan et al. 2015; danko et al. 2018; castaño-osorio et al., 2019) . although, still most of them are seemed to be slow immunity booster and require a strong adjuvant and longer immunisation to achieve full protection against each of the dengue serotype, which all make them ill-suited for universal vaccine licence . the dengue envelope (e) protein is composed of three ecto-domains, a membrane-proximal stem and a transmembrane anchor (klein et al. 2013 ). various crystal structures have shown that the ecto-domains are arranged into three antiparallel dimer on virus surface in a icosahedral symmetry, where ecto-domain i (ei) is located at the centre holding domain ii (edii) and iii (ediii) in two opposite sites in each of the monomer of a dimeric unit (fibriansah et al. 2015; kuhn et al. 2002; modis et al. 2004 ). most of the protective antibodies against dengue virus are identified to domain iii, and initially were thought to be a potential subunit vaccine target (murphy and whitehead 2011; sukupolvi-petty et al. 2010) . although, recombinant ediii domain injected by plasmid dna or purified from bacterial expression systems was found to be poorly immunogenic, and has shown low protective efficacy in animal model (guzman et al. 2010 ). on the other hand, edii has been reported as the dimerisation domain and consists of the most conserved fusion (fu) and bc loop (li et al. 2019; zhang et al. 2004 ). several studies have also shown that the conserved fu loop is highly immunogenic (lai et al. 2008; cherrier et al. 2009; smith et al. 2013) , and induces cross-reactive antibodies, of which some are crosstalk with adjacent bc loop (gallichotte et al., 2015; cherrier et al. 2009 ). moreover, during endocytosis, both of the loops are found to involve in tertiary conformation of membrane fusion (nayak et al. 2009; sukupolvi-petty et al. 2010) . therefore, it has been anticipated that both of the loops might have integrated role for boosting cross-neutralizing immunity. in this study, we aim to develop an alternative vaccine by combining fu and bc loop together in a single orf for production of a fusion antigen. although, the recombinant antigen in isolation tends to be poorly immunogenic in vivo; the use of potent immunomodulating compounds, fusion partner or suitable delivery systems improve specific immune response (higgins et al. 2007; garçon et al. 2011) . herein, we have expressed the recombinant fubc antigenic protein, optimised its large-scale purification protocol and finally evaluated its protein-specific immune response in balb/c mice. prior to the animal challenge, its secondary structure was checked by cd spectroscopy, and binding specificity was cross-checked with a characterised anti-fusion loop scfv antibody ). all of the male and female balb/c mice were obtained from national institute of nutrition, telangana, hyderabad, india and were maintained in standard light: dark (12:12) cycle with the supplement of adequate standard food, and water was provided from ad libitum. all of the animals were acclimated and randomly distributed into different experimental groups. furthermore, all the in vivo experiments were performed in accordance with the committee for the purpose of control and supervision on experiments on animals (cpcsea) guidelines and were approved by the south asian university institutional animal ethics committee (iaec) that was responsible for the care and use of laboratory animals. the dna sequence of fubc gene was retrieved from fusion (fu) and bc loop of denv serotype 2 envelope protein deposited in the protein data bank (pdb: 1oan). in order to construct stable and immunogenic protein, highly conserved fusion (fu) and bc loops and their neighbouring residues (amino acid residues 62 to 122) were selected by using antigen variability analyser (avana), pblast and multiple sequence alignment tool of ncbi. for convenient expression in bacteria, all of the oligos were codon optimised for e. coli, using codon optimisation tool of integrated dna technology. complete fubc gene was constructed by assembly pcr reactions using four 60 nucleotide long overlapping oligonucleotides. for cloning into pet28a expression vector, two unique restriction sites, ecori and xhoi, were also incorporated at the 5′ and 3′ ends respectively during the final amplification of fubc full-length gene using forward and reverse primers. initially, the full-length fubc gene was cloned into a ta cloning vector using instaclone kit (thermo scientific). positive clones were screened using x-gal bluewhite screening method and digested with ecori and xhoi restriction enzymes. the digested fubc gene was further sub-cloned into a pet28a expression vector. the recombinant plasmid (fubc + pet28a) was transformed into e. coli xl-10 gold for cloning, and subsequently into e. coli bl-21 rosetta (de3) for protein expression. the e. coli bl-21 (rosetta) cells carrying fubc gene in pet28a vector were grown overnight at 37°c in 10 ml lb broth (luria-bertani medium) containing 50 μg/ml kanamycin (sigma, usa). overnight grown 1 ml primary culture was used to inoculate kanamycin containing 100 ml secondary culture and was further incubated at 37°c. the incubated secondary culture was induced by 0.5 mm isopropyl β-d-1thiogalactopyranoside (iptg) when its od 600 reached at around 0.5, and the culture was grown for another 4 h at 37°c. the cells were harvested by centrifugation at 4000 rpm for 10 min. the resulting cell pellet was resuspended in lysis buffer containing 50 mm tris-hcl ph 8.0, 1 mm cacl 2 , with 0.5% triton x-100, lysozyme 0.1 mg/ml, 1 mm edta and 1 mm pmsf. then, the resuspended cells were kept on a rocker for an hour at room temperature, and sonication was done using 30% amplitude for five times 30 s on/off pulse. the lysed sample was separated into supernatant and pellet by centrifugation at 10,000 rpm for 10 min at 4°c. finally, fubc expression level in supernatant and pellet were checked on 15% sds-page. a major fraction of the fubc protein was observed in pellet after a number of efforts made to recover it in a soluble form. then, it was decided to recover soluble protein from pellet fraction. the resulting pellet protein was washed 4 to 5 times with te 50/20 (50 mm tris ph 8.0 and 20 mm edta 20) buffer to remove impurities and extra salts. the remaining pellet was re-solubilised using mild denaturing agent such as 4.0 m urea and 5% n-propanol along with pbs buffer (ph 8.1) and was centrifuged at 20,000 rpm for 10 min. the soluble protein fraction was initially purified by using ni-nta agarose beads and was confirmed by western blot using anti-his antibody. however, the purity and yield were not sufficient. therefore, soluble protein fraction was subjected to gel filtration in superose 6 10/300 column by using fast protein liquid chromatography (fplc) for largescale good quality protein production. the column was preequilibrated with pbs (50 mm phosphate buffer ph 7.4 and 150 mm nacl), and the protein sample was eluted with the same pbs buffer. 0.5 ml protein sample was injected in each run by using 0.5 ml loop at 0.4 ml/min flow rate. the elution profile of injected protein was followed by monitoring uv absorbance at 280 nm on the akta fplc system with u9-l uv monitor. different peaks greater than 20 mau were collected in fraction collector and checked using a 15% sds page. multiple runs of fplc were carried out, and the fraction containing fubc protein was pooled together. finally, fubc was concentrated and desalted by using 0.5 ml millipore-amicone filter with a cut-off of 3 kda. concentration of the purified fubc was also measured by using bca protein assay kit. in order to assure the proper folding of purified (> 95%) fubc protein, secondary structure was analysed by cd spectroscopy at far uv wavelength ranging from 180 to 260 nm. to achieve the best conformational reading, cd spectrum was obtained at the different protein and buffer concentrations, because the cd spectrum of a protein needs to be adequately intense for interpreting the data as the intensity of a cd spectrum directly relies on the protein and buffer concentration (kelly et al. 2005; miles and wallace 2006) . the cd spectra of pbs buffer and fubc protein samples at 0.1 mg/ml and 0.2 mg/ml concentrations (diluted in 50 mm and 10 mm pbs buffer, ph 7.4) were recorded at far uv spectra ranging from 200 to 280 nm with a step size of 1 nm to bandwidth 1 nm. the measurement was performed at room temperature (22°c), and the uv spectra were recorded for each sample with five scans. the baseline cd spectrum of the buffer was deducted from the spectrum containing the protein to yield the actual fubc protein cd spectrum. the mean residue ellipticity [θ] mrw at wavelength λ was quoted in units of degree cm 2 / dmol, and was calculated as [θ] mrw, λ = mrw × θλ/10 × d × c, where θ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm) and c is the concentration (g/ml). mrw is the mean residual weight for the peptide bond which is given as mrw = m/(n − 1); where m is the molecular mass of the polypeptide chain (in da), and n is the number of amino acids in the chain; the number of peptide bonds is n − 1. complete and incomplete freund's adjuvant (sigma, usa) was used for primary (day 0) and booster immunisation (days 7, 14 and 21) respectively. to prepare a primary dose of the immunogen, complete freund's adjuvant (fa) was mixed with equal volume of purified fubc (25 μg) in pbs and emulsified by vigorous vortex. similarly, three booster doses of immunogen were prepared with an equal volume of fubc protein (25 μg) and incomplete freund's adjuvant. all of the immunogens were prepared according to the protocol "immunization of mice" by maira-litrán, 2017. finally, emulsified fubc immunogens were checked by observing stable droplet on the water surface. healthy balb/c mice (10 weeks old, male and female) were randomly distributed into four different groups. each group was immunised with different antigen preparations. out of the four, two groups were immunised with fubc antigen: one group was injected with fubc protein with adjuvant and other was with fubc protein without adjuvant. rest of the two groups were immunised with fu and bc peptide: one group was injected with fu and bc peptides with adjuvant, and other was fu and bc peptide without adjuvant. each of the groups was also subdivided into male and female sets, and with each set, one adjuvant control was used replacing adjuvant with pbs buffer. the mice of each group were subcutaneously immunised with emulsified antigen preparations, first dose (day 0) with cfa and three subsequent booster doses (at days 7, 14 and 21) with ifa (according to protocol maira-litrán 2017). after 5 days of the final booster dose, blood samples were collected by retro-orbital cavity, and all the antisera isolated from blood were stored at − 80°c for further analysis. recombinant fubc protein-specific igg was measured by indirect elisa. for this, 96 well elisa plates were coated by incubating overnight at 4°c with fubc protein (1 μg/ well) in 50 mm sodium carbonate-bicarbonate buffer ph 9.5. blocking was done for 2 h with 2% bsa in pbst (pbs plus 0.05% tween 20). serum collected from immunised and control mice were incubated for 1 h at 37°c after making 7 double dilutions in pbst starting from 1/500 μl. subsequently, the elisa plate was washed once with pbst, and 100 μl of anti-mouse hrp-conjugated secondary antibody (1:1000 dilution in pbst) was added and incubated for 30 min at room temperature. then, the plate was washed three more times with pbst, and the binding reaction was developed by adding 200 μl/ well opd substrate (prepared in a phosphate-citrate buffer, 0.05 m, ph − 5.0 plus 0.03% h 2 o 2 ). the reaction was stopped by adding 50 μl of 2.5 m h 2 so 4 just after observing an optimum colour, and the absorbance was measured at 492-nm wavelength using biotek synergy ht microplate reader (winooski, vt). mean absorbance was plotted against anti-sera dilutions, and two-way anova was performed to find a statistically significant difference between two groups. comparison of the immune response in different groups of antisera was made by converting each elisa curve to linear equations (y = mx + c), and computing serum dilutions for a fixed elisa absorbance. the data are expressed as mean ± standard deviation (sd). p values of < 0.05 were considered statistically significant. to validate the immune response generated by recombinant fubc, collected mice anti-sera were allowed for spr interaction experiment with a autolab esprit instrument. similar to elisa, here also recombinant fubc protein of denv envelope was immobilised on a gold plate of spr device. for coupling of fubc protein on gold disc, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (edac) and nhydroxysuccinimide (nhs) were applied for activation. nhs activates the carboxymethyl groups by creating a highly reactive succinimide ester on the disc surface, which reacts with amine and other nucleophilic groups on proteins that subsequently help to bind the target protein on activated disc surface. ethanolamine was added to block the remaining activated carboxymethyl group. for the qualitative assay, all of the serum samples (diluted in running buffer) were applied at a flow rate of 20 μl/min over 2 min. in between injections, the surface of the sensor chip was regenerated by injecting 2 m nacl at the same flow rate for 15 s. in addition, surface coupling was done by using buffer (150 mm nacl, 3 mm edta, 0.005% surfactant p20 and 10 mm hepes-naoh) at ph 7.4, and the buffer containing 50 mm phosphate and 150 mm nacl at ph 7.1 was used for sample running. initially, a series of serum dilutions were applied as analytes to find an optimum refractive index. it was observed that the refractive index at 1:500 dilution was within the detection limit among the series of serial dilutions (fig. 6a) . therefore, 1:500 dilution of different experimental samples was applied as standard for further comparative analysis between fubc immune response with and without freund's adjuvant. serum control (the serum collected before immunisation) sample with the identical dilution was also applied to find the basal (non-specific) immune response of serum. finally, refractive index was then analysed from association and dissociation curve of the spr sensorgram. the fubc synthetic gene sequence was deposited in genbank database with accession number mn781186. generally, dengue envelope (e) folded into three distinct domains (designated by domain i, ii and iii), membrane proximal stem and a transmembrane anchor (klein et al. 2013) . throughout these structural element, four highly conserved regions have been identified by in silico sequence analysis, and two of them were found in the domain ii with a very less informational entropy . further studies have revealed that these conserved regions are the part of previously characterised flavivirus fusion (fu) and bc loop (kuhn et al. 2002) . during the process of endocytosis, this hydrophobic fusion loop remains buried at the dimer interface in the prefusion state and forms cluster into larger hydrophobic surface at one end to form trimer at later state that finally initiates membrane fusion (klein et al. 2013 ). in addition to this structural property, fu and bc regions consist some of the most potent antigenic epitopes that were identified by denv neutralising conformation sensitive anti-denv hmabs (goncalvez et al. 2007; costin et al. 2013; yamanaka et al. 2013) . therefore, these two conserved fu and bc loops have been selected to design a recombinant fubc antigenic protein for the development of dengue subunit vaccine. in this study, an alignment of the domain ii amino acid sequences of denv1-4 envelope proteins spanning residues from 61 to 120 was used to find optimum conserved sequence for the development of fubc fusion protein (fig. 1a) . the structural details of truncated fubc protein compare to whole envelope in dengue and fu, and bc peptides were also analysed by modelling each of their three dimensional structure. it reveals that the presence of three anti-parallel β-strands and one disulphide linkage play a crucial role in preserving the three dimensional conformations of truncated fubc protein same as in original denv envelope protein (fig. 1b, c) . ironically, due to the absence of anti-parallel β-strands and a single protein frame of fu and bc loops, these two separately expressed peptides fail to form similar three-dimensional conformation as in original denv envelop (fig. 1c) . the electrostatic and solvent accessible areas of fubc truncated protein are also comparable with original dengue envelope. hence, it is speculated that the recombinant fubc protein would be an alternative vaccine target of whole dengue envelope. fu loop is shown in orange and bc loop is shown in yellow. b in denv envelope, the fu and bc loops are present at the end of domain ii and are linked by the di-sulphide linkage that holds both loops together to form a stable structure that can also work as an epitope. c when the structure of fubc is compared to original denv envelope, it is plausible that the presence of three anti-parallel β-strands present in fubc protein (same as denv protein) plays a crucial role in maintaining the three dimensional conformation of fubc protein as it is in denv protein. another major factor that plays in conformation of fu and bc loops is the disulphide linkage between the two highly conserved loop in the fubc protein that ensures the same conformation of denv envelope as it is shown in b and c, and d due to the lack of anti-parallel β-strands and same protein frame while these peptides are expressed separately, it is unlike to form di-sulphide linkage and fold in the same conformation as reside in denv envelope and fubc proteins for in vitro synthesis of short antigenic protein, a recombinant fubc gene was constructed by assembly pcr using overlapping oligonucleotides, designed from fu and bc loop encoding dna sequences of dengue envelope. therefore, full length of fubc gene was amplified by using 5 and 3 end primers flanked by ecori and xhoi restriction sites. the fubc gene was then cloned into a ta cloning vector, and the clones were screened by colony pcr using fubc gene specific end primers. two positive clones were then confirmed by restriction digestion using ecori and xhoi enzymes (fig. s1a-d) . the digested and purified fubc insert gene was further sub-cloned into pet28a expression vector, and the positive clones were confirmed by restriction digestion (fig. s2b) and sanger sequencing. primarily, the recombinant fubc protein expression was observed only in pellet fraction; there was no such fubc equivalent protein band in supernatant fraction, while they were separated on sds page (fig. s2c) . therefore, fubc expression level was checked further by lowering growth temperature and iptg concentration, but no significant change was noticed in supernatant fraction. chaperone-assisted folding system did not help remarkably to express the fubc recombinant protein in the soluble form. therefore, the insoluble pellet fraction of fubc protein was further utilised in mild solubilisation process to recover soluble fubc protein. initially, the recovered soluble fubc protein fraction was purified by using ni-nta agarose beads, and the purified protein band was confirmed by western blot analysis using anti-his tag monoclonal antibody (fig. s3) . although, ni-nta affinity chromatography has not revealed good purification quality and the yield was also not sufficient. furthermore, gel filtration chromatography was used to recover good quality large-scale recombinant protein, and single distinct peak greater than 20 mau was observed at approximately 20.57 ml position for all of injected fubc protein samples (fig. 2a) . after pooling together, the peak fractions were separated on 15% sds-page, and single bright band was observed at molecular weight 11.5 kda (fig. 2b) . originally, the fu and bc loop of dengue envelope is composed of three anti-parallel β-sheets. and the formation of recombinant fubc secondary structure was characterised here by negative bending at 208 nm and 222 nm wavelength (kumagai et al. 2017) . the cd spectrum of recombinant fubc protein has showed significantly decreased spectral peaks around 222 and 208 nm that signify the formation of β-sheet (fig. 3 ). in addition, it was noticed that the acquired cd spectrum of fubc at a protein concentration of 0.1 mg/ml in 10 mm pbs buffer was adequately intense (fig. 2) . therefore, it can be inferred from fig. 3 (lower panel) that fubc at concentrations of 0.2 mg/ml and 0.1 mg/ml in 10 mm pbs retained the best globular folded state as compared to other conditions. immune response to the purified fubc protein in balb/c mice from indirect elisa, it was observed that the serum igg levels in both male and female groups treated with fubc proteins were significantly higher than those treated with only adjuvant and pbs control (fig. 4a, b) . in addition, the response with only fubc recombinant protein (without adjuvant) in the female group was also observed higher than their male counterpart group (fig. 4b) . all of the elisa data were also found statistically significant by two-way anova (table s1) . similarly, by spr assay, high level immune response was also observed for serum injected with recombinant fubc protein along with adjuvant, and without any adjuvant, the response was also higher than the control (serum with pbs only) (fig. 6b ). in post-dengue infection, most of the circulating antibodies are non-neutralizing and found to be raised against dengue envelope e protein and the prm protein (wahala and de silva 2011) . due to the absence of highly specific neutralising antibodies in secondary infections, cross-reactive nonneutralising antibodies usually enhance the dengue severity (de alwis et al. 2014 ). according to the revised dengue case classification (denco), 2 to 3% of secondary infections with another serotype causes life threating dengue with warning signs (dws+) and severe dengue (sd). it was reported that serotype-cross-reactive non-neutralising antibodies enhance the entry of dengue genome into fc receptor-bearing monocyte cells and promote disease severity by a process known as antibody-depended enhancement (ade) flipse et al. 2013) . that means nature of the antibody response to denv is most likely to play a major role in defining disease outcome. therefore, it is predictable that antibodies that recognise specific neutralizing epitopes help in virus clearance and reduce symptoms; however, antibodies that recognise non-neutralising epitopes lead to more severe forms of disease like dws+/sd. hence, there is an urgent need for an advanced vaccine which could generate highly specific and cross-neutralising antibody (costin et al. 2013 ). recently, several attempts have been made towards the development of a potent dengue vaccine. the most advanced candidate, dengvaxia (cyd-tdv), was licenced in some of the dengue endemic countries (imai and ferguson 2018) . however, it was revealed risky in children or naïve dengue patient with severe infection as they were vaccinated (arredondo-garcía et al. 2018) . therefore, considering safety issues, production of recombinant subunit vaccine with efficient immune protective properties is looking attractive (govindarajan et al. 2015) . meanwhile, an admixture of four live attenuated recombinant dengue vaccine tv003/tv005 have completed phase iii clinical trial and licenced to several manufacturers including butantan, vabiotech and merk (whitehead et al. 2017) . moreover, some recombinant tetravalent vaccines (e.g. den-80e, tvdv) expressing the prm and e genes of each of the four denv serotypes from plasmid dna, have already completed phase i clinical trial (govindarajan et al. 2015; danko et al. 2018 ). it has also been shown that the recombinant dengue envelope domain iii can inhibit dengue infectivity, and induce dengue-neutralising immunoglobulin in mice (hermida et al. 2006) . in addition, a number of antibodies which were raised in mice and fig. 2 purification of recombinant fubc protein by size exclusion chromatography. a fplc chromatogram of fubc protein expressed in e. coli inclusion bodies. three distinct peaks at 19.88 ml, 20.57 ml and 22.57 ml position were collected separately. b gel image of unpurified and purified fubc protein sample separated on 15% sds-page. lanes 1 and 2 represent the un-purified sample and lanes 3 and 4 represent purified protein sample collected from peak at position 20.57 ml fraction chimpanzees against dengue domain ii fusion loop were found cross-reactive to other flaviviruses (goncalvez et al. 2004; stiasny et al. 2006) . although, the hmabs specific to dengue domain ii fusion loop were not found equally effective on other flaviviruses including wnv, yfv and other denv strain (costin et al. 2013) . therefore, it was conjectured that adjacent to the fusion loop, additional contact residues of original domain ii surface structure might be involved to raise cross-neutralizing antibodies. several studies have identified that adjacent to the fusion loop, another similar loop exists in most of the flaviviral (e.g. wnv, tbe, jev, yfv) envelope (fibriansah and lok, 2016; li et al. 2019) . our previous bioinformatics studies have also shown that the envelope of all denv serotype consists of four conserved regions (> 90%), and two of them were found in domain ii, in which one is fusion loop (fu) with more than 99% conservation and another is its nearby bc loop (fig. 1a ) . therefore, these two highly conserved loops were targeted in this study for the development of a fusion protein. by using reverse dna technology, we have previously shown the structural integrity ( fig. 1b-d) and binding specificity of fubc protein with an anti-fusion loop scfv antibody, derived from dengue and wnv-specific mab e53 (rodenhuis-zybert et al. 2011; rathore et al. 2019) . the very early challenge of subunit vaccine design is to produce large quantities of functional protein. hereby, we have successfully optimised the expression and purification methods for the production of large-scale high-quality recombinant fubc protein. the e. coli expression system was used here, and the recombinant protein was found to be expressed in higher quantity, though most of the protein was extracted initially in pellet fraction. no significant change was noticed by altering regular growth temperature and inducer concentration. therefore, we have utilised the mild-solubilisation methodology to recover soluble fubc protein from insoluble pellet protein. for purification, firstly, we have used convenient ninta affinity purification method, and by adjusting the lysis and elution buffer, we were able to purify recombinant fubc protein to some extent but the quality and quantity was not sufficient. finally, to scale up the quality protein production, size exclusion chromatography was used in akta fplc system. to confirm the recombinant protein expression, simple western blot was performed by using anti-his monoclonal antibody as primary. furthermore, in vitro structural integrity and functional authenticity of the experimental fubc protein were also verified by cd spectroscopy (fig. 3) and indirect elisa (fig. 4) . however, most of the hmabs identified earlier from dengue patient serum were predominantly cross-reactive and recognise epitopes containing highly conserved residues at the fu loop of domain ii (lai et al. 2008 ). however, having such sequence homology in fu loop of all flaviviruses, these hmabs are non-neutralizing against heterologous serotypes (lai et al. 2008; smith et al. 2013) and thus was found to be responsible for ade in animals (goncalvez et al. 2007 ). previously, it has also been stated that this scenario might happen due to the cryptic nature and poor accessibility of the fusion loop epitope on the surface of the mature virion (lai et al. 2008; cherrier et al. 2009 ). however, in addition to the partially exposed fusion loop on immature flaviviruses, some neutralizing hmabs were also found to bind with bc loop (costin et al. 2013) . therefore, it is suggestive that along with conserved fusion loop, adjacent less conserved linker sequence and bc loop might be required to be exposed as a whole to generate effective cross-neutralizing immunity. our current mouse immunisation experiment also supports this idea as it was observed that the antibody response to full length fubc protein was stronger than the response elicited by the fu and bc peptides in separate (figs. 4 and 5) . moreover, without freund's adjuvant, recombinant fubc protein was found immunogenic, and the response in female mice was stronger than in male (figs. 4 and 5) . these observations were also symmetrical with other studies where it was stated that female mice have a better immune response due to the higher number of leukocytes occupying the naive peritoneal and pleural cavities, and also have a number of t-and blymphocytes as well as macrophages (terres et al. 1968; weinstein et al. 1984; klein et al. 2015) . therefore, we have used female mice serum samples for further qualitative elisa and spr test to quantify immune response of recombinant fubc protein. it was noticed that igg antibody response to fu, and bc peptides were significantly lower than the response observed to fubc recombinant protein, and the immune response achieved to fu and bc peptide with freund's adjuvant was significantly higher as compared to the peptides without adjuvant and serum control (fig. 5) . two-way anova was also performed to analyse the significance of igg immune responses between fubc + fa and fubc without fa; fu, bc peptide + fa and fu, bc peptide without fa. from f values and p values obtained by anova test, it can be interpreted that the difference between fubc protein with freund's adjuvant and fu, bc peptide with freund's adjuvant were significantly higher than protein and peptides injected without freund's adjuvant (table s2) . also, the igg response to recombinant fubc protein was recorded better than fu and bc peptides in both cases either with or without adjuvant. in addition, the p value of igg immune response between fubc protein with fa and without fa was found insignificant (p = 0.1740 > 0.05) that suggests that fubc without any adjuvant is sufficient to elicit significant immune response (table s2) . consistently, it was observed by spr experiment that the refractive index of fubc + fa was significantly higher than fubc + without fa and serum control (fig. 6b) . since, the newly developed fubc recombinant protein expresses vastly in e. coli and induces significant immune response in mice, it might be a good agent of dengue subunit vaccine development. due to the presence of highly conserved fusion loop epitope, overlapping less conserved linker sequences and also bc epitope, this fusion protein might induce cross-neutralisation immunity against heterologous dengue serotypes. though, still a lot of investigations, like the evaluation of a proper adjuvant to induce robust immune response, the memory immune response generated against the fubc protein both by humoral and cell-mediated immunity, and whether this memory response will provide protection against a secondary encounter with denv, are required before going clinical trial. nevertheless, the production process and immune response of this fusion protein would provide new insight for the development of dengue subunit vaccine. four-year safety follow-up of the tetravalent dengue vaccine efficacy randomized controlled trials in asia and latin america the global distribution and burden of dengue refining the global spatial limits of dengue virus transmission by evidencebased consensus current status of vaccines against dengue virus. dengue fever -a resilient threat in the face of innovation intechopen book chapter 9 structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody the development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop development of dengue dna vaccines safety and immunogenicity of a tetravalent dengue dna vaccine administered with a cationic lipid-based adjuvant in a phase 1 clinical trial dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera phase i randomized study of a tetravalent dengue purified inactivated vaccine in healthy adults from puerto rico development and clinical evaluation of multiple investigational monovalent denv vaccines to identify components for inclusion in a live attenuated tetravalent denv vaccine dengue virus cryo-em structure of an antibody that neutralizes dengue virus type 2 by locking e protein dimers the development of therapeutic antibodies against dengue virus molecular mechanisms involved in antibody-dependent enhancement of dengue virus infection in humans rs 2015 a new quaternary structure epitope on dengue virus serotype 2 is the target of durable type-specific neutralizing antibodies development of a dengue vaccine and its use in pregnant women 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vaccine: considerations for serosurvey design how to study proteins by circular dichroism structure of a dengue virus envelope protein late-stage fusion intermediate sex-based differences in immune function and responses to vaccination structure of dengue virus: implications for flavivirus organization, maturation, and fusion going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain ii potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes immunogenicity and efficacy of flagellin-envelope fusion dengue vaccines in mice and monkeys immunization of mice dengue virus pathogenesis: an integrated view synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics structure of the dengue virus envelope protein after membrane fusion immune response to dengue virus and prospects for a vaccine crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion dengue vaccination: a more balanced approach is needed designing antibody against highly conserved region of dengue envelope protein by in silico screening of scfv mutant library a fusionloop antibody enhances the infectious properties of immature flavivirus particles evaluation of scfv protein recovery from e. coli by in vitro refolding and mild solubilization process the potent and broadly neutralizing human dengue virus-specific monoclonal antibody 1c19 reveals a unique cross-reactive epitope on the bc loop of domain ii of the envelope protein cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2 a quantitative difference in the immune response between male and female mice the human antibody response to dengue virus infection sex-associated differences in the regulation of immune responses controlled by the mhc of the mouse in a randomized trial, the live attenuated tetravalent dengue vaccine tv003 is well-tolerated and highly immunogenic insubjects with flavivirus exposure prior to vaccination a mouse monoclonal antibody against dengue virus type 1 mochizuki strain targeting envelope protein domain ii and displaying strongly neutralizing but not enhancing activity publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-010991-fp8hljbq authors: rather, shabeer ahmad; sharma, sukesh chander; mahmood, akhtar title: antibodies generated against dextransucrase exhibit potential anticariostatic properties in streptococcus mutans date: 2020-01-03 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-10327-x sha: doc_id: 10991 cord_uid: fp8hljbq streptococcus mutans is a common principal causative agent of dental caries. in this communication, we describe that the antibodies raised against purified dextransucrase effectively inhibited the growth of s. mutans. the purified enzyme showed 58-fold enrichment, 17.5% yield and a specific activity of 3.96 units/mg protein. purified igg fraction of the antibody showed significant affinity with the antigenic protein. immunotritation of the enzyme with dextransucrase antibody showed a gradual increase in inhibition of dextransucrase activity. the growth of s. mutans was also inhibited by 85% in the presence of 28 μg of igg fraction of the antibody. antibodies also impaired glucosyltransferase activity (72.8%) and biofilm formation by 92.6% in s. mutans. western blot analysis revealed no cross reactivity with the various tissues of mice, rat, rabbit and humans. dot blot analysis showed little reactivity with lactobacillus acidophilus and staphylococcus aureus and there was no reactivity with other bacterial strains like enterococcus faecalis, escherichia coli and salmonella typhimurium. these findings suggest that antibody raised against dextransucrase exhibit inhibitory effects on the growth of s. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent. dental caries is globally the most prevalent disease of mankind causing demineralisation of tooth enamel by acids produced by the oral bacteria (yang et al. 2017; talbman and smith 1974) . it is a multifactorial biofilm-mediated disorder, which is initiated by dysbiosis in the biofilm complex, where certain bacteria take the dominance over others in the oral flora. it has been shown that many bacteria are involved in the genesis of caries formation at different stages. among these streptococcus mutans is the primary causative agent of dental caries (alam et al. 2018 ). s. mutans is an acidogenic and aciduric microorganism, well characterised to produce dental caries (loesche 1996) . it has the ability to generate large quantities of extracellular polysaccharides (dextrans) from sucrose under the action of dextransucrase (ec.2.4.1.5)/glucosyltransferases (gtfs) accompanied with its adhesion and acid-producing activities (lynch et al. 2013 ). these dextrans help in the attachment of microbe to tooth surface leading to infection (kuramitsu 1974) . a number of compounds such as penicillin, cationic agents (chlorhexidine), plant products (polyphenols, flavonoids, anionic agents (sodium dodecyl sulphate) and non-ionic agents (triclosan) have been used for the prevention of dental caries by inhibiting growth and adherence of these cariogenic bacteria to the tooth surface (jarvinen et al. 1993; chen and wang 2010) . but these organisms are either resistant to them (alam et al. 2018; bhattacharya et al. 2003) or the drugs exhibit side effects (craig 1998) . studies on the prevention of cariogenicity have also focussed on antibody production and hence vaccine development from adaptive immunity. for vaccine development, attention was paid on the purified antigens involved in the pathogenesis of dental caries for the development of potentially safer vaccines, which may reduce the viability of bacteria in the saliva, impairing the surface adhesion and inhibiting the metabolically active enzymes involved in caries formation (chen and wang 2010) . many surface molecules of s. mutans such as lipoteichoic acid, glucosyltransferases (gtfs), antigen a (a 29-kda protein antigen), antigen c (a 70-kda protein antigen), antigen d (a 13-kda protein antigen), agi/ii (a 190-kda protein), agiii (39-kda protein), gbp (glucan-binding protein) (kruger 2004) , gtfb (kim et al. 2012 ) and dna-based active vaccines, synthetic peptides and mucosal adjuvants (heat-labile enterotoxins (hlt) from vibrio cholera (lt-i) or escherichia coli (lt-ii), bupivacaine, chitosan) have attracted great attention for passive immunisation in the prevention of the dental caries (yan 2013; chen and wang 2010; fan et al. 2002; xu et al. 2007; alam et al. 2018) . fusion vaccines (pgja-p/vax and pgjg/gac/vax) encoding pac and glu of s. mutans were also tested in gnobiotic animals (kt et al. 2013 ) and flagellin-pac fusion protein (kf-rpac) was also tested in rats for anticaries vaccine (bao et al. 2015) . antibodies raised against recombinant form of substrate binding component of the phosphate uptake system (rpsts) of s. mutans have shown protective response against caries formation (ferreira et al. 2016) . cao et al. (2016) found no significant effect of specific s-iga antibody on caries formation. yang et al. (2019) developed the intranasal cold-adapted influenza vaccine, which was limited by the large size of the vector than s. mutans epitope, this resulted in memory immune response thus reducing the duration and intensity of exogenous antigens. among the various proteins of s. mutans, dextransucrase has an essential role in the synthesis of glucan from sucrose, thus play a crucial role in the pathogenesis of the caries (talbman and smith 1974) . strategies of using adaptive immunity for the generation of antibodies against various purified molecules of s. mutans have shown encouraging results related to dental caries protection, but were limited by the cross-reactive epitopes against human heart and skeleton muscle tissues as detected by indirect immunofluorescence and crossed immunoelectrophoresis (kt et al. 2013) . hajishengallis and michalek (1999) however reported that glucosyltransferase when tested for cross reactivity with human heart tissue showed negative results. in the present study, we have tried to evaluate the effect of anti-dextransucrase antibodies on caries formation by using purified dextransucrase as the antigen from s. mutans. the evaluation of anti-dextransucrase antibodies demonstrated that they inhibited several of the cariogenic characteristics of s. mutans, thus have the potential for development of anticaries agent. some of these results are described in this communication. the study was approved by central animal ethics committee panjab university chandigarh (iaec no. pu/iaec/s/16/52). s. mutans strains mtcc-890 were grown in brain heart infusion (bhi) broth, supplemented with 1% dextrose, 1% peptone, 0.29% glucose, 0.25% sodium hydrogen phosphate and 0.05% nacl (ph 7.4) to late-exponential phase at 37°c. s. oralis was grown in tryptic soy agar (tsa) (himedia, mumbai, india). e. coli, s. aureus and s. typhimurium were grown in nutrient agar at 37°c and l. acidophilus was grown in mrs media (sisco research laboratories pvt. ltd., new mumbai, india) . all studies relating to dextransucrase were carried out using mtcc-890 strain of s. mutans. antigenic protein dextransucrase was purified from culture supernatant of s. mutans mtcc-890 by ammonium sulphate precipitation followed by sephadex g-200 column chromatography. the pooled fractions from column chromatography were treated with peg-400. after centrifugation at 15,000 g to separate the dextransucrase fraction, the pellet was dissolved in 10 mm sodium maleate buffer (ph 6.8) dialysed overnight using dialysis membrane-135 (himedia, mumbai, india) (liu et al. 2010) . the dialysate obtained served as the antigen. except otherwise stated all procedures were carried out at 4°c (goyal 2015) . concentrations of the purified proteins were detected by bradford protein assay (campion et al. 2017) . the purity of dextransucrase protein was assessed by sds-page (laemmli 1970 ). dextransucrase activity was assayed by using standard reaction mixture containing 0.05 m sodium maleate buffer (ph 6.8), 0.1 m sucrose, in total volume of 0.5 ml. after incubation for 30 min at 37°c, the samples were assayed for glucose using the glucostat kit (reckon diagnostic p. ltd.). the results were expressed in enzyme units per milligram of protein. one unit of dextransucrase activity was defined as the amount of enzyme required to release 1 μmol of glucose per min under standard assay conditions. new zealand white rabbits 6-8 months old were purchased from animal facility of indian institute of microbial technology (imtech), chandigarh, india. animals were kept in the animal facility of the department of biochemistry panjab university chandigarh under specific-pathogen-free (spf) conditions. they were housed in a metallic cage and were provided with food and water ad libitum. animal studies were performed in compliance with the guidelines of committee for the purpose of control and supervision of experiments on animals (cpcsea). six-month old new zealand white rabbits were immunised subcutaneously with purified dextransucrase protein (500 mg/ml) emulsified in freund's complete adjuvant (f5881, sigma, usa). the injection sites were clipped and disinfected with 70% alcohol before injections were made. the injections of around 250 μl each were given at four different sites in the limbs. blood sample of 2 ml was taken before injection. serum was collected from blood by centrifugation for analysis. booster injections were given post immunisation after 3 weeks and repeated again after 4 weeks and 6 weeks using the antigen emulsified with freund's incomplete adjuvant. test bleeds were taken after 8 days of each booster and checked for serum antibody using dot blot assay. sera was collected 2 weeks after injecting the booster immunisation dose for the analysis of dextransucrase antibody. presence of serum antibody raised against dextransucrase antibody was checked by dot blot analysis (vera-cabrera et al. 1999 ). dot blot assay was performed by spotting 2 μl of samples slowly to minimise area onto the nitrocellulose membrane at the centre of the grid drawn by a pencil. the membrane was allowed to dry at room temperature and non-specific sites were blocked by soaking in 5% skim milk in phosphate buffered saline tween-20 (pbst) for 2 h at 37°c. the membrane was washed three times in phosphate buffered saline (pbs) (nacl 137 mm, kcl 2.7 mm, na 2 hpo 4 10 mm, kh 2 po 4 1.8 mm) ph 7.4 and overnight incubated at 4°c in 1:50 dilution of rabbit serum in 5% skimmed milk. after washing three times with pbs, it was incubated with hrp-conjugated goat anti-rabbit secondary antibody diluted 1:6000 for 1 h at 37°c. excess secondary antibody was washed three times 10 min each with pbs developed by enhanced chemiluminescence system (ecl) and observed under gel-doc uvtech for fluorescence. samples positive for serum showed black dots and negative showed clear dots. to localise the target protein in the cells of s. mutans, immunocytochemistry was performed by culturing bacterial cells on glass coverslips in 6-well culture plates (greiner bio-one india pvt. ltd.) at 37°c for 36 h. after 36-h of growth, the media were aspirated and cells were washed gently with pbs. cells were fixed in fixation buffer (4% formaldehyde and 0.2% glutaraldehyde) for 2 h. after fixation, cells were rinsed twice gently with pbs and blocking solution 3% bsa (bovine serum albumin) was added and incubated for 90 min. blocking solution was aspirated and cells rinsed with pbs. primary antibody raised against the target protein diluted in blocking buffer was added and incubated overnight at 4°c. primary antibody was aspirated from wells and washed 3times with pbs and fitc (fluorescein isothiocyanate)-tagged secondary antibody (sigma-aldrich, usa) diluted in blocking solution was added and incubated for 1 h at room temperature in the dark. after incubation with secondary antibody, the cells were washed with pbs, counterstained with dapi (4, 6-diamidino-2-phenylindole) sigma-aldrich, usa, diluted 1:5000 in pbs for 1 min and washed with distilled water. the glass coverslips were mounted using mounting medium cells-side down onto microscope slides. the coverslips were observed under confocal microscope. the experiment were repeated in triplicates. serum collected from immunised rabbits was analysed for dextransucrase-specific antibody titre by elisa. flat-bottom 96-well elisa plates (nest biotech co. ltd., china) were coated with 10 μg of antigenic protein in 0.1 m bicarbonate buffer (ph 9) and incubated for overnight at 4°c. wells were washed three times with pbs-0.02% tween-20 to remove unbound proteins and blocked in 3% blocking buffer for 2 h. after washing wells with pbs-0.02% tween-20, rabbit serum samples were serially double diluted, loaded in the wells (first well with 1:100 dilution of antibody) and incubated for 2 h at room temperature. subsequently, the wells were washed three times with pbs-0.02% tween 20 which was followed by addition of hrp (horseradish peroxidase)-conjugated goat anti-rabbit igg (dilution 1:6000) antibody (genei, bangalore, india) and incubated for 1 h at 37°c. the liquid was aspirated and wells washed three times with pbs-0.02% tween 20. tetramethyl benzidine (tmb) solution 100 μl/well was added for 5-10 min followed by addition of tmb stop solution (conc. h 2 so 4 ) 100 μl to each well. absorbance was read at 450 nm using microplate reader (synergy/hi biotek, india). the experiment was repeated three times. values of the dilution giving an o.d reading that was two times of background were determined as the titre. igg fraction from rabbit serum was purified by affinity chromatography by using ready to use prepacked protein a-sepharose column (bio vision, usa) with a binding capacity of ≥ 16 mg rabbit igg/ml protein a-sepharose with a flow rate of 2.0 ml/min. the column was equilibrated with pbs as binding buffer (nacl 137 mm, in 20 mm na-k phosphate buffer ph 8.0). the serum sample was diluted with binding buffer in the ratio of 1:1 volumes, mixed well and applied to the column. the flow through was collected and reapplied 4times to the column. the column was washed 4-5 times with the × 5 volume of binding buffer and the antibodies were eluted with the elution buffer (0.1 m citric acid, ph 2.75). the fractions were collected and neutralised immediately by adding 100 μl of 1 m tris ph 9.0 per ml of eluate. protein concentration was assayed by measuring the absorbance at 280 nm. the minimum inhibitory concentrations (mic) of serum antibody against s. mutans was determined by microdilution method as described by hasan et al. (2014) . s. mutans was grown in basal bhi medium with increasing concentrations of serum antibody ranging from 0 to 40 μg/ml at 37°c for 24 h under aseptic conditions. the mic was the lowest concentration that inhibited the visible growth of the bacteria. however, the bacterial colonies could not be counted as the growth was essentially negligible. all the determinations were performed in triplicates. immunotritation of dextransucrase was performed using standard reaction mixture containing 0.05 m sodium maleate buffer (ph 6.8), 0.1 m sucrose, in total volume of 0.5 ml in presence of increasing concentrations of igg (0-70 μg). the reaction mixture was incubated at 37°c for 30 min and assayed for glucose using the glucostat kit (reckon diagnostic p. ltd.). the results were expressed in enzyme units/mg protein. the tissue samples were placed in ice cold lysis buffer (ripa buffer sigma-aldrich, usa) (product code r 0278) which contained 50 mm tris-hcl, ph 8.0, with 150 mm sodium chloride, 1.0% igepal ca-630 (np-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate and phosphatase inhibitor cocktails (sigma-aldrich, usa). the tissues were lysed by homogeniser and incubated at 4°c overnight and centrifuged at 15,000 g at 4°c for 20 min. the supernatant was stored and quantified for protein concentration. the protein lysate was run on 10% sds-page and transferred on to nitrocellulose membrane. western blot analysis was done following the method described by mahmood et al. (1993) . primary antibody was diluted in the blocking buffer in the ratio of 1:500 (v/v). hrp-conjugated goat anti-rabbit igg (dilution 1:7000 v/v) was used as the secondary antibody. the membrane was developed by enhanced chemiluminescence (ecl) and the bands were analysed by imagej software for densitometry. to examine the inhibitory effect of serum antibody on biofilm formation by s. mutans, biofilm microplate assay was performed by standard protocol (merritt et al. 2005) . s. mutans were grown in bhi (brain heart infusion) supplemented with 5% sucrose with a concentration of 3-5 × 10 6 cfu/ml or culture optical density adjusted to 0.015 at 600 nm (od 600 ) in 96-well microtitre plates precoated with saliva to mimic the conditions of oral cavity. sub mic of serum antibody were added and wells without serum igg served as control. after incubation at 37°c for 20 h, the planktonic cells were decanted and adherent cells were stained with 0.1% crystal violet solution for 15 min. the wells were washed with pbs twice, dried and the bound dye was extracted by adding 33% acetic acid to completely destain the wells. biofilm formation was measured by measuring the absorbance of resuspended solution at 590 nm on a biotek microplate reader. cross reactivity of dextransucrase antibody with mammalian tissues was evaluated by western blot analysis. proteins from different tissues such as the liver, lungs, spleen, heart, kidney and gall bladder of rat, mice, rabbit and human were resolved on 10% sds-page. samples containing 30 μg of protein mixed with loading dye and heated for 5 min were loaded in each well and the molecular weight marker was run in a separate lane so as to identify the band of interest during western blotting. resolved unstained proteins were transferred onto nitrocellulose membrane and detected as described above. cross reactivity of anti-dextransuccrase antibody was evaluated by using dot blot method. the cell extracts of various bacterial strains were spotted on the nitrocellulose membrane, allowed to dry and placed in 5% skimmed milk for 2 h to block non-specific sites. after blocking, the membrane was incubated in serum antibody (dilution 1:500) raised against dextransucrase from s. mutans for 1 h at 37°c followed by incubation in hrp-conjugated goat anti-rabbit secondary antibody diluted 1:6000 for 1 h at 37°c. the affinity of serum antibody was observed by developing the membrane by chemiluminescence using gel-doc uvtech. transferase activity of dextransucrase enzyme was assayed by modified method of mukasa et al. (1979) . an adequate preparation of enzyme solution containing 0.1 m phosphate buffer (ph 6.5), 41.7 mm sucrose, 34.3 μm dextran and 0.02% sodium azide with and without purified anti-dextransucrase antibody was incubated for 15 h at 37°c. the mixture was centrifuged at 17,000g for 15 min to collect water-insoluble glucan formed. the water soluble glucan was precipitated with 75% ethanol and collected by centrifugation at 17,000g. the glucans formation was determined by phenol sulphuric acid method (dubois et al. 1956 ) in which glucose is dehydrated to hydroxyl methyl furfural which forms a yellow brown coloured product with phenol with an absorption maxima at 490 nm. enzyme activity was expressed in units/mg protein. one unit of glucosyltransferase activity was defined as the amount of the enzyme catalysing the transfer of 1 μmole of the glucose to glucan per min. purification of the antigenic protein dextransucrase from s. mutans the culture supernatant of s. mutans grown at 37°c for 36 h was used as protein source. after 55% ammonium sulphate precipitation, the dextransuccrase activity was enriched by 14-fold with 56% recovery. enzyme preparation after fractionation on sephadrexg-200 chromatography followed by treatment with peg-400 the dextransucrase activity was purified 58-fold with recovery of 17.5%. the analysis of the enzyme purification on sds-page using 10% gel showed that the purified enzyme after peg-400 fractionation gave the single protein band corresponding to 160 kda as shown in fig. 1 . antibodies were raised against the purified dextransucrase by injecting to rabbits subcutaneously as mentioned in material and methods. the serum collected from rabbit after booster immunisation was screened for the presence of the dextransucrase antibody by dot blot analysis and confocal microscopy. three spots of purified protein, culture supernatant of s. mutans and bsa respectively were made on the nitrocellulose membrane and incubated with the dextransucrase antibody. the antigen antibody complex was detected using hrp-conjugated secondary goat anti-rabbit antibody. as shown in fig. 2a , dextransucrase antibody showed strong affinity with the purified protein, culture supernatant of s. mutans demonstrating presence of antibody generated against dextransucrase. however, no reactivity was seen with the bsa which served as the control. to further validate the generation of antibody against dextransucrase, immunofluorescence by confocal microscopy of s. mutans cells for the affinity of the target protein was carried out. as shown in fig. 2b , green fluorescence of s. mutans cells depicted significant immunoreactivity of the antibody against dextransucrase thus confirming the generation of specific antibody against antigenic protein dextransucrase. the antibody titre of serum from immunised rabbit with dextransucrase from s. mutans was determined using elisa. antigenic protein dextransucrase of 10 μg coated to wells of 96-well plate and reacted with different dilutions of antidextransucrase serum. wells with no coating of antigen served as blank, wells treated only with primary antibody but no secondary antibody, served as primary control and wells treated with secondary antibody only served as secondary control. the linear regression was used to calculate r 2 that is linear correlation between serum dilution and od. the results of elisa as shown in fig. 2c demonstrated that the affinity decreased with dilution. the affinity of antisera was detected at a dilution of 1:64000 and the non-coated wells showed estimate of similar to cut-off i.e. negative results. purification and characterisation of igg serum igg fraction was purified from immunised rabbit serum using affinity protein a-sepharose column (bio vision milpitas, usa). igg was detected only in first four fractions collected in affinity column chromatography by measuring the absorbance at 280 nm using uvspectrophotometer which had concentrations of 0.299 mg/ml, 4.00 mg/ml, 0.703 mg/ml and 0.0919 mg/ml respectively. the fractions containing igg were analysed on 10% sds-page which showed two clear bands of igg having molecular weight of 50 kda and 25 kda against a prestained molecular weight marker of 175 kda (pink plus gene direx) fig. 3a . the fractions containing purified igg were pooled and tested by immunoblots using the samples of purified dextransucrase, culture supernatant and bsa as control. the antigen antibody complex was detected by using hrp-conjugated secondary goat anti-rabbit antibody (1:7000) dilution by enhanced chemiluminescence (ecl) system. the results are shown in fig. 3b . the effect of dextransucrase antibody on the growth of s. mutans was studied under in vitro conditions. s. mutans cells were grown in presence of different amounts of antibody ranging from 0 to 40 μg/ml for 20 h at 37°c. as shown in fig. 4 , 28 μg/ml concentration of igg was found to be the minimum inhibitory concentration (mic) which inhibited the growth of s. mutans to nearly 85% of the control indicating significant antimicrobial activity. to further study the inhibitory effect of purified antibody on the enzyme activity of dextransucrase, immunotritation was evaluated. increasing concentrations of the igg from 0 to 70 μg/ml to enzyme protein were added and enzyme activity was assayed after 30 min at 37°c. as shown in table 1 , there was a progressive increase in the percentage inhibition from 2.58 to 18.22 as compared with the control. thus, antibody against dextransucrase exhibited inhibitory effect on the activity of dextransucrase isolated from s. mutans. cross reactivity of the antibody against various mammalian tissues was studied by western blot analysis. protein samples of the liver, heart, spleen, kidney of mice, rat and liver, kidney of rabbit were analysed by western blot analysis to evaluate the cross reactivity of purified antibody with mammalian tissues fig. 5a . evaluation of western blot analysis showed that the anti-dextransucrase antibody in the dilution of 1:500 did not cross react with any of the mammalian tissues tested; however, there was a strong reactivity of the antidextransucrase antibody with the s. mutans-derived dextransucrase used as control. western blot analysis of the human heart, liver and gall bladder was also tested for cross reactivity with anti-dextransucrase antibody. as shown in fig fig. 4 growth inhibition of s. mutans by purified igg under in vitro conditions. the cell culture was treated with different amounts of igg ranging from (0-40 μg protein) per culture tube. bacterial growth was determined by measuring o.d at 600 nm after 20-h incubation at 37°c. the values are mean ± sd, n = 3 5b, there was no cross reactivity with these organs but showed a strong reactivity with the s. mutans-derived dextransucrase which served as the positive control. immunoblot analysis of cell extracts from gram-positive and gram-negative bacterial strains such as s. oralis, l. acidophilus, s. aureus, e. faecalis, e. coli and s. typhimurium respectively was also studied to check the reactivity of these strains with dextransucrase antibody. as shown in fig. 6 , there was little reactivity of antibody raised against dextransucrase with l. acidophillus and s. aureus and no reactivity with other bacterial strains confirming the specificity of the antibody against s. mutans. the anti-biofilm formation tendency of antibody raised against dextransucrase from s. mutans was investigated by crystal violet assay. the s. mutans cells were grown under in vitro conditions treated with 20 μg purified igg. the assay was carried out in triplicates in three sets of samples as blank, treated and control in triplicates in 96-well plate and incubated for 20 h at 37°c. as shown in fig. 7 , the value of crystal violet assay of antibody treated sample was 0.090 as compared with control 1.232 as quantified by photometric estimation at o.d 590 . biofilm formation in antibody treated sample was reduced by 92.6% as compared with the control and statistically significant reduction in biofilm formation was observed at 28 μg/ml of serum igg. dextransucrase antibody showed significant inhibition of glucosyltransferase activity in s. mutans dextransucrase have both hydrolytic and transferase activity. it catalyses the transfer of glucosyl residues from sucrose to dextrans. these results showed that enzyme activity was 0.212 units/mg protein in control which was reduced to 0.0576 units/mg protein in the presence of 30 μg of igg, indicating a decrease of 72.8% in glucosyltransferase activity under these conditions (fig. 8 ). s. mutans is the well-established etiological agent in the pathogenesis of dental caries. the virulence traits by which infection is established includes biofilm formation by sucrose dependent and independent means, adherence, aciduricity, the purified enzyme was treated with different concentrations of antidextransucrase igg ranging from 0 to 70 μg. the enzyme activity was determined after incubation of 30 min at 37°c as described under materials and methods. values are mean ± sd, n = 3 fig. 5 assay of cross reactivity of dextransucrase antibody with various mammalian tissues (a). immunoblots of tissues samples from liver, heart, spleen and kidney of rat (i), mice (ii) and rabbit (iii). purified dextransuccrase was used as the control. the tissue protein was resolved on sds-page by using 10% gels, transferred onto nitrocellulose membrane and detected by western blot analysis using purified dextransucrase antibody (1:500) dilution followed by hrpconjugated secondary goat anti-rabbit antibody (1:7000) dilution. the blots were developed by ecl system. b the reactivity of dextransucrase antibody with tissues proteins of the heart, liver and gall bladder from humans by immunoblot analysis. purified dextransuccrase was used as control. acidogenicity and hydrophobic interactions (hasan et al. 2014; ferreira et al. 2016) . immune intervention against dental caries has been pursued for the last many years to develop a potent and effective vaccine that may help to combat this chronic infectious disease (zhang 2013) . several cell surface substances of s. mutans that have been used for vaccine preparation include glucosyltransferases, adhesins and glucan-binding proteins (giasuddin et al. 2017) . efforts are being made to raise antibodies against various s. mutans antigens and to examine their effect on caries formation (hajishengallis and michalek 1999) . in the previous studies, whole cells of the mutans streptococci were used as possible vaccine for the dental caries. surface substances of s. mutans including glucosyltransferases (gtfs), lipoteichoic acid, antigen a (29-kda protein), antigen c (70-kda protein), antigen d (13-kda protein), agi/ii (190-kda protein), agiii (39-kda protein) and gbp (glucan-binding proteins (koga et al. 1995; kuramitsu 1993) play crucial role in pathogen and host interaction. kim et al. (2012) have also reported the formation monoclonal antibody against clonal fragment of glucosyltransferase b in s. mutans gs-5 and showed its inhibitory activity against the enzyme. thus, these surface molecules have attracted much attention for the development of vaccine against dental caries. in the present study, we generated antibodies against dextransucrase principally involved in the metabolism of sucrose which is the main substrate of s. mutans in establishing the dental caries. dextransucrase was purified and antibodies against it were raised in rabbits. the raised antibodies were checked for affinity with dextransucrase using dot blot assay which showed significant reactivity and further validated by immunofluorescence using confocal microscopy which confirmed specific binding of dextransucrase antibody with the cells of s. mutans. dextransucrase helps s. mutans in the fig. 7 a effect of dextransucrase antibody on biofilm formation by s. mutans under in vitro conditions. the cells were treated with antibody (28 μg). data are mean ± sd n = 3. the bar represent p value < 0.001 compared with control. blank = only culture media, positive control = s. mutans culture without dextransucrase antibody, test = s. mutans culture with dextransucrase antibody. b biofilm formation assay performed in 96-well plate using crystal violet staining of biofilm. fig. 8 effect of anti-dextransucrase antibody on the glucosyltransferase activity of dextransucrase enzyme. transferase activity was determined by measuring the amount of sugar content after treating the enzyme dextransucrase with antibody. standard = assay system contained glucose with no dextran, no enzyme and no antibody. control = enzyme + dextran test = enzyme + dextran + antibody. data are mean ± sd, n = 3 metabolism of sucrose for its growth and producing waterinsoluble glucans with mixed α-1, 3 and α-1, 6 linkages (moye et al. 2014) which are involved in the attachment of s. mutans on the tooth surfaces and in the biofilm formation (bowen and koo 2011) leading to caries formation. immunotitration of dextransucrase with purified dextransucrase antibody showed a progressive increase in percentage inhibition of enzyme activity, although the antibody titre was high, presumably due to its polyclonal nature. also it was observed that the growth of s. mutans in the presence of different concentrations of antibody under in vitro conditions was inhibited, which correlated with the inhibitory effect on dextransucrase activity; however, the values were quantitatively distinct. this is in agreement to the studies of culshaw et al. (2007) on anticaries protection after immunisation of animals with intact gtf. transferase activity of dextransucrase also showed a significant reduction in presence of anti-dextransuccrase antibody which may help in reducing glucan formation. biofilm formation proceeds with the attachment of bacterial cells to the tooth surface and subsequent formation of multilayered cell clusters (challan et al. 2006; wen and burne 2002) . the biofilm is stabilised when the bacterial cells in the three-dimensional structure grow vertically and get connected through extracellular polysaccharides (ansari et al. 2017) produced by the metabolism of sucrose with the action of dextransucrase. song et al. (2015) have shown that biofilm formation is strongly dependent upon the surface adhesion and hydrophobicity of the material. more recently, nilsson et al. (2019) have shown that oxidative stress plays a role in the antibiotic tolerance of s. mutans biofilm. in our study, the efficacy of dextransucrase antibody on biofilm formation was studied using crystal violet microplate assay in which cells were grown in saliva coated wells for 20 h which demonstrated that there was 92.6% reduction in the biofilm formation in antibody-treated samples as compared with control suggesting that the dextransucrase antibody has inhibited enzymatic activity of dextransucrase and thus disrupted the bacterial adherence and subsequent aggregation of cell clusters leading to biofilm formation. although various types of vaccines have been developed and tried for caries prevention like whole cell vaccine, conjugate vaccine, synthetic vaccine and dna vaccine etc. and showed promising results for the protection against dental caries, however, it was reported that antibodies generated showed cross reactivity with human heart tissues and skeleton muscle tissues (ayakawa et al. 1985) . this was further confirmed by other investigators and these immunologically cross-reactive polypeptides were found in the cell membrane of s. mutans (giasuddin et al. 2017) . subunit vaccines have also been tried to overcome the cross reactivity but were reported having less immunogenicity (zhang 2013) . it is also assumed that besides s. mutans, other oral bacteria also play a role in the pathogenesis of dental caries although the mechanism is not established. nevertheless, the inhibitory effect of dextransucrase antibodies against several cariogenic factors of s. mutans may be helpful in formulating strategies to combat the disease (alam et al. 2018) . present data showed that the antibodies generated did not cross react with protein extracts from the liver, heart, lungs and kidneys of mice, rat and rabbit by western blot analysis. protein extracts from the human heart, liver and gall bladder were also checked for cross reactivity with the dextransucrase antibodies by western blot analysis which revealed negative results. we further investigated the cross reactivity of dextransucrase antibody with various bacterial strains and this specificity was tested by using total protein extract of various gram-positive and gram-negative bacterial strains. protein extracts form s. mutans, s. oralis, l. acidophilus, s. aureus, e. faecalis, s. typhimurium and e. coli were evaluated for cross reactivity by immunoblot analysis and the results have shown little reactivity with l. acidophilus and s. aureus other than s. mutans and there was no reactivity with the other bacterial strains. the reactivity with the l. acidophilus and s. aureus suggests a similar epitope recognised between two strains. since the dextransucrase antibody recognised the similar epitopes on the two oral strains, it can be predicted that anti-dextransucrase antibodies raised against s. mutans could recognise other species of the oral cavity, thereby strengthens the support to dextransucrase as a candidate vaccine against dental caries and shared epitopes between cariogenic strains can show better efficacy in clinical trials. thus, the data highlights the importance of antibody against dextransucrase which has inhibitory effect on the growth of s. mutans and worked effectively against biofilm formation. our preliminary observations also indicated that dextransucrase antibodies when added to the growth media inhibited acid production and reduced hydrophobicity of s. mutans (results not shown) which further corroborated the anticariogenic properties of the dextransucrase antibodies. moreover, examination of various animal and human tissues showed no cross reactivity with dextransucrase antibody which implies that they may have no harmful physiological effects in the humans, thus may prove a promising antigen for developing anticariogenic agent. further studies to characterise the cariogenic effects of antibody against dextransucrase are underway to ascertain these findings. the study was approved by central animal ethics committee panjab university chandigarh and experiments were performed in compliance with the guidelines of committee for the purpose of control and supervision of experiments on animals (cpcsea). human tissues were obtained from the histopathology department of postgraduate institute of medical education and research chandigarh in compliance with the standards of institutional ethical committee. methods statement authors declare that all experiments and procedures performed with animals were performed in accordance with relevant guidelines and regulations of the institutional animal ethics committee of panjab university, chandigarh, india. synthetic antigen-binding fragments (fabs) against s. mutans and s. sobrinus inhibit caries formation anti-biofilm activity of a self-aggregating peptide against streptococcus mutans immunochemistry of the streptococcus mutans bht cell membrane: detection of determinants cross-reactive with human heart tissue flagellin-pac fusion protein inhibits progression of established caries inhibition of streptococcus mutans and other oral streptococci by hop biology of streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms protein quantitation and analysis of purity immunogenicity and prediction of epitopic region of antigen ag i/ii and glucosyltransferase from streptococcus mutans assessment of the roles of luxs, s-ribosyl homocysteine, and auto inducer 2 in cell attachment during biofilm formation by listeria monocytogenes egd-e novel technologies for the prevention and treatment of dental caries: a patent survey antimicrobial resistance-danger signs all around immunogenic and protective potential of mutans streptococcal glucosyltransferase peptide constructs selected by major histocompatibility complex class ii allele binding colorimetric method for determination of sugars and related substances a dna vaccine encoding a cell-surface protein antigen of streptococcus mutans protects gnotobiotic rats from caries sublingual immunization with the phosphate-binding-protein (psts) educes oral colonization by streptococcus mutans dental caries vaccine availability: challenges for the 21st century current status of a mucosal vaccine against dental caries inhibition of major virulence pathways of streptococcus mutans by quercitrin and deoxynojirimycin: a synergistic approach of infection control in vitro susceptibility of streptococcus mutans to chlorhexidine and six other antimicrobial agents a monoclonal antibody specific to glucosyltransferase b of streptococcus mutans gs-5 and its glucosyltransferase inhibitory efficiency surface proteins of streptococcus mutans passive immunisation against oral pathogen. from the division of clinical immunology at the department of laboratory medicine and center for oral biology at novum, institute of odontology. karolinska institute dental caries vaccine -a 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cariogenic pathogen, streptococcus mutans publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements shabeer ahmad rather is a recipient of senior research fellowship from icmr new delhi. we thank to mr. naveed parvaiz of department of zoology panjab university, chandigarh, india, for providing tissue samples of mice and rat and dr. chetan for rabbit tissues samples. we appreciate the help of professor praveen rishi of department of microbiology panjab university, chandigarh, india, for providing bacterial strains s. aureus, l. acidophilus, s. typhimurium, e. coli and e. faecalis. authors' contributions s.a.r wrote the main manuscript and performed experiments, a.m contributed in concept design and/or analysis and interpretation of data and revising it critically for important intellectual content and s.c.s provided culture facilities and reviewed the article for important inputs. the article was reviewed for final submission by all authors.competing interests the authors declare that they have no conflict of interest. key: cord-012437-2r6egrca authors: lenz, markus; enright, anne marie; o’flaherty, vincent; van aelst, adriaan c.; lens, piet n. l. title: bioaugmentation of uasb reactors with immobilized sulfurospirillum barnesii for simultaneous selenate and nitrate removal date: 2009-05-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-009-1915-x sha: doc_id: 12437 cord_uid: 2r6egrca whole-cell immobilization of selenate-respiring sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 µg se × l(−1)) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). gel-immobilized s. barnesii cells were used to inoculate a mesophilic (30°c) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (cod) × l(−1) day(−1). selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 µg se × l(−1) was obtained. scanning electron microscopy with energy dispersive x-ray (sem–edx) analysis revealed spherical bioprecipitates of ≤2 µm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. to validate the bioaugmentation success under microbial competition, gel cubes with immobilized s. barnesii cells were added to an upflow anaerobic sludge bed (uasb) reactor, resulting in earlier selenate (24 hydraulic retention times (hrts)) and sulfate (44 hrts) removal and higher nitrate/nitrite removal efficiencies compared to a non-bioaugmented control reactor. s. barnesii was efficiently immobilized inside the uasb bioreactors as the selenate-reducing activity was maintained during long-term operation (58 days), and molecular analysis showed that s. barnesii was present in both the sludge bed and the effluent. this demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation. electronic supplementary material: the online version of this article (doi:10.1007/s00253-009-1915-x) contains supplementary material, which is available to authorized users. excess of nitrate (1,500 times) and sulfate (200 times). gelimmobilized s. barnesii cells were used to inoculate a mesophilic (30°c) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (cod)×l −1 day −1 . selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39µg se×l −1 was obtained. scanning electron microscopy with energy dispersive x-ray (sem-edx) analysis revealed spherical bioprecipitates of ≤2µm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. to validate the bioaugmentation success under microbial competition, gel cubes with immobilized s. barnesii cells were added to an upflow anaerobic sludge bed (uasb) reactor, resulting in earlier selenate (24 hydraulic retention times (hrts)) and sulfate (44 hrts) removal and higher nitrate/nitrite removal efficiencies compared to a nonbioaugmented control reactor. s. barnesii was efficiently immobilized inside the uasb bioreactors as the selenatereducing activity was maintained during long-term operation (58 days), and molecular analysis showed that s. barnesii was present in both the sludge bed and the effluent. this demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation. selenium contamination of soil and water is a problem of global importance. although the ecotoxicological effects of selenium poisoning were documented in the western us already 30 years ago, a cost-effective solution to the problem has not yet been found and the threat to wildlife persists (hamilton 2004; presser and luoma 2007; wu 2004) . biological reduction processes are considered a promising approach to decontaminate large quantities of such agricultural drainage waters polluted with nitrate (∼3.5 mm) and low concentrations of soluble selenium oxyanions (selenite and selenate, ∼3.5µm; oremland et al. 1999 ) because of their high selectivity toward the targeted oxyanions (lenz et al. 2008b) . denitrifying microorganisms have been proposed as key biocatalysts for the treatment of this type of (waste)waters due to the selenium oxyanion-reducing ability of both membrane bound and periplasmatic nitrate reductases (sabaty et al. 2001) . the specific activities for selenate reduction by nitrate reductases are, however, 15 to 518 times lower (watts et al. 2005 ) and the affinity constants (k m ) 2.3 times higher for selenate compared to nitrate reduction (sabaty et al. 2001) . consequently, selenate is reduced by these enzymatic systems at low nitrate concentrations only . one approach to achieve these sufficiently low nitrate levels, thus enabling selenate reduction, is a two-compartment reactor system, implemented in, e.g. the algal-bacterial selenium reduction system (amweg et al. 2003) . in the first compartment, nitrate levels are reduced in a high rate pond by microalgal assimilation, whereas selenate is biologically reduced to <100µg/l by bacteria in anaerobic ponds of the second compartment (green et al. 2003) . however, space prerequisites for these reduction steps are high, and further treatment steps (dissolved air flotation and slow sand filtration) are required to remove algal biomass and remaining selenium particles prior to the discharge of the effluent to the environment (amweg et al. 2003) . selenate-respiring microorganisms contain specific selenate reductases not competitively inhibited by nitrate (schröder 1997) . this specificity of the selenate-reducing enzymatic systems is further underlined by the fact that selenium-respiring organisms can also completely reduce selenate in the presence of a high molar excess of the structural analog sulfate (lenz et al. 2008b ). since so specific for selenate, selenium-respiring organisms may offer an alternative to the currently applied two-step process. however, bioaugmentation by simple addition of cell cultures is typically limited by wash-out and outcompetition of the inoculated culture by the endogenous microorganisms (el fantroussi and agathos 2005) . immobilization in gel has been suggested to counteract these limitations, yet the performance has not been tested in continuous experiments so far (morita et al. 2007; tucker et al. 1998) . sulfurospirillum barnesii is a particularly promising inoculum as it can respire a variety of substrates (including both selenium oxyanions, nitrate, and nitrite), produces elemental selenium as end-product of selenium respiration, and is non-pathogenic stolz et al. 1999) . in order to assess the performance of bioreactors in continuous operation, the inoculation with immobilized selenium-respiring microorganisms was investigated in the present study. to validate the applicability of gel immobilization under microbial competition, one upflow anaerobic sludge bed (uasb) reactor was inoculated with both immobilized bacteria and anaerobic granular sludge. the selenate removal efficiency was evaluated in comparison to a non-bioaugmented uasb reactor. the chemical composition of selenium bioprecipitates was investigated by sem-edx. in addition, denaturing gradient gel electrophoresis (dgge) and nucleotide sequencing were used to evaluate the immobilization success. source of biomass s. barnesii (strain 10660) was obtained from the german collection of microorganisms and cell cultures (dsmz, braunschweig, germany). anaerobic granular sludge originated from a full-scale uasb reactor treating paper mill wastewater (industriewater eerbeek b.v., eerbeek, the netherlands). for s. barnesii cell immobilization experiments, bacterial cells were pregrown in medium prepared according to dsmz, containing nitrate as electron acceptor and lactate as electron donor (20 mm each, see electronic supplementary material for complete medium composition). during bioreactor operation, oxygen-free synthetic wastewater containing macronutrients, micronutrients (lenz et al. 2006) , and a vitamin solution (according to dmsz medium for s. barnesii) was used. the medium was buffered at ph=7.0 (±0.1) using a 40 mm phosphate buffer. cells were harvested in the exponential growth phase by centrifugation of 20 ml cell suspension at 11,000×g for 20 min (iec cl31r multispeed centrifuge, thermo scientific, breda, the netherlands). the pellet was resuspended in 500µl of s. barnesii medium, and 125µl of this suspension was added to 50 ml of polymerizing gel and stirred gently. gelling conditions were used according to tucker et al. (1998) , but using different ratios of n,n′methylenebisacrylamide (mbaa) and acrylamide (aa). the gel was poured into sterile plastic beds of 8 cm×8 cm× 4 mm. after hardening, the gel was cut with a sterile scalpel into cubes of 4×4×4 mm. all immobilization steps were conducted under n 2 atmosphere in a glove box. characterization of the s. barnesii cubes the effect of the gelling conditions on the fracture stress of the gel cubes was determined by the strength needed to burst a 1-cm 3 gel cube at the brittle point, measured by a penetrometer (overload dynamics s900, overload dynamics, schiedam, the netherlands). the influence of the gel composition on the selenate reduction efficiency was studied in 125-ml batch bottles containing ten gel cubes with immobilized s. barnesii cells (total of 16.1µg biomass dry weight) in 50 ml of synthetic wastewater. selenate was added from a concentrated stock solution to a final concentration of 100µm. the batch bottles were subsequently flushed with a sterile stream of n 2 and incubated at 30°c on a horizontal shaker at 120 rpm. the bioreactors (0.46 l working volume) were operated under mesophilic conditions (30±1°c) and a hydraulic retention time (hrt) of 6 h as described previously (lenz et al. 2008b ). to avoid nitrate, selenate, or sulfate bioconversion in the storage vessels, influents were composed of three different streams, fed in the same ratios to the reactor: (1) solution of selenate, sulfate, nitrate, macro-/micronutrients, and vitamins, (2) lactic acid dissolved in phosphate buffer, and (3) dilution water. three bioreactors (r1-r3) were inoculated as follows: r1 received 25 gel cubes (1:30, mbaa/aa). r2 was operated as a regular uasb reactor (lenz et al. 2008b ) and inoculated with 100 g wet weight [37.4×g volatile suspended solids (vss) −1 l −1 ] of anaerobic granular sludge. r3 received both cubes and sludge in the same quantities used to inoculate r1 and r2, respectively. lactate was used as sole electron donor at an influent concentration of 13 mm (12 mol of electrons per mole lactate), resulting in an organic loading rate of 5 g cod× l −1 day −1 , corresponding to a specific organic loading rate of 134 mg cod×gvss −1 day −1 . nitrate was supplemented to the reactors as main electron acceptor at an influent concentration of 15 mm (5 mol of electrons per mole of nitrate reduced) in period i (days0 to 24) and period iii (days43 to 58), whereas no nitrate was supplied in period ii (days25 to 42). sulfate and selenate (accepting 8 and 6 mol of electrons during reduction to sulfide and elemental selenium, respectively) were supplemented at influent concentrations of 2 mm and 10µm, respectively, during the whole reactor operation. for sem, samples were fixed for 1 h in an aqueous glutaraldehyde solution (2.5%), rinsed with water, and dried either in a n 2 stream or in a series of ethanol (lenz et al. 2008b) . samples were then fixed to a brass sample holder with carbon adhesive tabs (electron microscopy sciences, hatfield, usa) and coated with 5 nm platinum by magnetron sputtering. specimens were analyzed with a field emission scanning electron microscope (jeol 6300 f, tokyo, japan). edx (inca energy, oxford instruments analytical, high wycombe, england) was performed at a voltage of 15 kv and a working distance of 15 mm. total genomic dna was extracted from the r2 and r3 biomass and an unfiltered effluent sample of r1 obtained at the conclusion of the trial (day58) using a dneasy® plant mini kit (qiagen, germany). extracted dna was visualized by uv excitation as previously described (enright et al. 2007 ). partial bacterial 16s rrna genes were amplified with the forward primer 341f and reverse primer 517r, a 40-base pair gc-clamp was attached to the 5′ terminus of the forward primer (muyzer et al. 1993) . the pcr reaction mixture and pcr conditions are described in the electronic supplementary material. polyacrylamide gels were prepared with denaturing gradients ranging from 30% to 70% denaturant (100% denaturant=7 m urea+40% formamide), and loaded with 20μl of the respective gc-clamped pcr products (muyzer et al. 1993) . gels were run at 65°c and 75 v for 16 h. following this, gels were stained/destained (10 min, respectively) and photographed on a uv transillumination table. bands of interest were excised from the dgge gels using a sterile scalpel blade, suspended in 50μl of sterile water, and stored at room temperature for 6 h to facilitate the elution of dna. both the pcr and gc-clamped pcr product analysis was repeated up to five consecutive times, in order to insure the presence of a single isolated band. pcrs were performed under the conditions described above, but without gc clamps attached to the forward primers. sequences were determined using a capillary sequencer (mwg biotech, germany) and aligned with 16s rrna gene sequences retrieved from the ribosomal database project (rdp; maidak et al. 1996) . sequences were then aligned to previously deposited sequences, downloaded from the rdp website, using clustalx (thompson et al. 1997) . the phylogenetic inference package paup* 4.0b8 was used for all phylogenetic analysis (swofford 2001) , using the kimura-2 parameter correction (kimura 1980; saitou and nei 1987) and partial bacterial 16s rrna gene sequences deposited in the genbank database (accession numbers in electronic supplementary material). selenate, selenite, nitrate, nitrite, and sulfate were determined by ion chromatography as described previously (lenz et al. 2006) . the dissolved sulfide concentration of the effluent was determined colorimetrically (dr. lange, lyw653, germany). total dissolved selenium (se dis ) was determined by inductively coupled plasma-optical emission spectroscopy (detection limit 10µg se×l −1 ) after filtration using a 0.45-µm pore size syringe filter (whatman, hertogenbosch, the netherlands). volatile fatty acids (vfas) and biogas composition were determined by gas chromatography (weijma et al. 2000) . gels containing mbaa/aa in a ratio of 1:30 had the highest fracture stress (table 1) . higher aa concentrations resulted in hard, shattering gels, whereas lower concentrations caused incomplete gelling. furthermore, the selenate removal rates in the 1:30 mbaa/aa gels were 14% and 41% higher compared to gels with a ratio of 1:20 and 1:40, respectively. consequently, the 1:30 gel cubes were used to inoculate r1 and r3. during the start-up (period i), low selenate and se dis removal efficiencies were observed in all three reactors (fig. 1a-c) . r3 showed the highest selenate reduction efficiency of the three reactors at the end of period i (20% on day 22). although selenate was removed by the bioreactor, total selenium analysis showed a wash-out of undetermined selenium species with the effluent (4% of the influent selenium). nitrate was removed completely by r1 and r2 within 8 and 6 days of operation, respectively, whereas a complete removal was achieved in r3 already 20 and 12 hrts earlier (fig. 2a-c) . in r2, complete nitrate removal was not sustained, and nitrate followed nitrite accumulation in the effluent after 13 days of operation (fig. 2b) . sulfate was not removed in any of the reactors in period i (fig. 2d-f ). the cod removal efficiency exceeded 82% and 87% throughout most of period i in r1 and r3, respectively, after a start-up period of approximately 6 days ( fig. 2g and i) . when nitrate removal was low in r2 (9.2 mm nitrate+0.7 mm nitrite in effluent, fig. 2b) , a slight accumulation of acetate (2.7 mm) was observed, resulting in a reduced cod removal efficiency of 54%. effect of bioaugmentation on reactor performance in the absence of nitrate upon omitting nitrate from the feed (period ii), r1 and r3 reacted with an immediate increase in both selenate and dissolved selenium (se dis ) removal efficiencies ( fig. 1a and c) . comparative selenium removal efficiencies were achieved in r2 only after a delay of 5 days (20 hrt; fig. 1b) . the se dis removal efficiency was lower than the selenate removal efficiency in all three reactors with maximal differences of 37%, 34%, and 43% in r1 to r3, respectively. toward the end of period ii, the difference became smaller in all three reactors. the lowest se dis effluent concentration was 39µg se×l −1 in r1, while r2 and r3 reduced se dis less efficiently (120 and 174µg se× l −1 , respectively). immediately upon the transition from period i to ii, the sulfate removal efficiency increased in the bioaugmented reactors (r1 and r3, fig. 2d and f) , while this occurred in fig. 2e ). the highest amounts of dissolved sulfide accumulated in r3 (1.2 mm, fig. 2f ), while 41% less dissolved sulfide was formed in r1. cod removal efficiencies were generally low in period ii compared to period i and iii (fig. 2g to h) . propionate was the main vfa accumulating in the r2 and r3 effluent, while propionate and acetate accumulated in the effluent of r1 (fig. 2g) . when resuming the nitrate feed (period iii), the selenate and se dis removal efficiency immediately decreased in r2 and r3 ( fig. 1b and c) , whereas this decrease occurred with a delay of 2 days of operation (8 hrt) in r1. upon termination of the reactor operation, an almost complete selenate removal was achieved in r2 (<97%), while r3 removed only 56% selenate. due to a blockage of the nutrient line, only lactate and dilution water was fed to r1 subsequently to day51. thus, selenate and se dis removal efficiencies could not be determined until the end of period iii. again, se dis was less efficiently removed compared to selenate, with a difference of up to 59% (r2) and 64% (r3). se dis even washed out of r3 in period iii (fig. 1c) . the application of a 0.1µm filter subsequently to filtration with a pore size of 0.45µm increased the se dis removal efficiency in r2 by 11% to 48% (period iii, data not shown). immediately upon resuming the nitrate feed, high nitrate/ nitrite removal efficiencies were achieved that exceeded 91% in both r2 and r3 (fig. 2b and c) , while sulfate removal efficiencies dropped upon transition to period iii ( fig. 2d to f) . the cod removal efficiency was high (>82%) throughout the whole period iii in both r2 and r3 ( fig. 2h and i) . r1 accumulated both acetate and propionate in the effluent, resulting in no net-cod removal upon the end of the reactor operation (fig. 2g) . during batch incubation of immobilized s. barnesii with selenate, a red-colored precipitate was first observed within the cubes and subsequently in the whole batch medium (fig. 3a) . sem analysis of the gel cubes following the batch experiments showed that the gel cube surface was entirely covered with selenium precipitates (fig. 3b) , whereas the inside of the cube contained fewer, but larger selenium precipitates (fig. 3c) . the edx maps show these consisted mainly of selenium, with smaller contributions of sulfur (scan s1, fig. 3c and d) . the gel cubes obtained from r1 upon termination of the reactor operation were entirely crusted by calcium and phosphorous containing precipitates, as demonstrated by the edx surface scan (spectrum s2, fig. 3e and h) . the cross-sections showed flower-like structures up to 70 µm from the edge of the cube ( fig. 3f and g) , mainly consisting of calcium and phosphorous (spectrum s3, fig. 3h ). the sludge bed volume of r2 and r3 more than doubled (∼220 ml final volume), and a color change from dark black to light gray with slime embedding the granules was observed during the reactor operation, but the granular character of the sludge remained. selenium accumulated in the sludge granules (and the embedding slime) of r2 and r3 up to 1067 (r2) and 1194µg se× gvss −1 , respectively. in r1, low amounts of white flocks formed that were loosely deposited on top of the cube bed. sulfurospirillum-like species were detected in the r1 unfiltered effluent and the r2 and r3 biomass samples obtained at the trial conclusion (b4-fj499345, figs. 4 and 5). furthermore, a selenium-reducing organism (selenatereducing bacterium tsa) was also detected in the r2 and r3 biomass samples (b20-fj499361, figs. 4 and 5 ). in addition, members of the closely related genus (i.e., klebsiella) previously shown to reduce selenate were indicated in the samples of r1 (b5-fj499346, figs. 4 and 5) . i ii iii i ii iii i ii iii i ii iii i ii iii i ii this study shows that gel immobilization can be used to immobilize both microorganisms and their precipitation products (fig. 3b and c) , i.e. elemental selenium, and sustain microbial reductive activity under long term (58 days) reactor operation (fig. 1a) . dgge and sequencing of the excised bands demonstrated that biomass was seeded to the reactor liquid and the sludge granules as a sulfurospirillum species were found in the effluent of r1 and the sludge of r3 after 58 days of operation (b4-fj499345, fig. 5) . thus, immobilization in gels is promising for applications requiring a continuous release of biomass (boon et al. 2002) , superseding wash-out or out-competition in bioaugmentation. for example, bioaugmentation of uasb reactors with sulfate reducers, so far unsuccessful due to the use of suspended cultures as bioaugmentation inoculum (vallero et al. 2004) , might be a future application. immobilization was achieved in non-biodegradable polyacrylamide gels (leenen et al. 1996) , which is important when considering full-scale applications. members of the genus klebsiella (klebsiella oxytoca, b5-fj499346, fig. 5 ) have been described to reduce selenate (zhang et al. 2008) and might contribute to the selenate removal in r1. proliferation of further microorganisms (additional bands in fig. 4) can also explain sulfate reduction in r1, as s. barnesii is not capable to use sulfate as terminal electron acceptor. although nitrate/nitrite concentrations were reduced to <1µm, selenate was not reduced at the start-up of the reactor (fig. 1a) . when incubating s. barnesii under high excess of nitrate (5 mm) to selenate (50µm) with lactate as electron donor, oremland et al. (1999) observed selenate reduction at a strongly decreased rate (factor >48) compared to incubations without nitrate. consequently, the incomplete removal efficiencies observed in period i of r1 and r3 can be explained by a kinetic limitation, i.e., the reactor medium is continuously replaced, resulting in a hydraulic retention too short to completely remove selenate. omitting nitrate from the feed consequently increases selenate reduction activity (during transition to period ii). the increase in selenate-reducing activity can be determined as 0.097 (r1) and 0.099µm selenate×µgs. barnesii initial −1 day −2 (r3) by fitting the increase in selenate removal efficiency linearly. during the first 20 (r1) and 22 days (r2+3) of reactor operation, no sulfate reduction was observed, thus no sulfide was available to chemically precipitate biogenically formed selenite (hockin and gadd 2003) . consequently, selenate was completely reduced to elemental selenium in period i. during later reactor operation, biogenically formed sulfide was present in all three reactors in molar excess to selenate (fig. 2) , which might have precipitated potentially formed selenite completely. the fact that se dis and selenate removal efficiencies in r3 were lower compared to r2 in period iii can be due to sulfide toxicity (lenz et al. 2008b ) as dissolved sulfide concentrations had build up to higher levels (1.2 mm) in period ii. fig. 1b) due to the proliferation of a selenium-respiring specialist (b20-fj499361, fig. 5) . thus, such reactors can be applied as an alternative to a two-step denitrifyingselenate-reducing process (green et al. 2003) or the bioaugmentation with selenium-respiring organisms. proliferation of a selenium-respiring population in anaerobic granular sludge of the same origin was also observed when it was operated under methanogenic conditions (lenz et al. fig. 3 a immobilized s. barnesii cells during production of elemental red selenium (0, 48, and 168 h) and sem pictures of batch cubes (b, c) with edx mapping (d) of selected area (s1) containing precipitates. surface (e) and cross-section (f, g) of a cube sampled after 58 days of reactor operation with (h) surface scan of s2 and of precipitate within the cube 2008b), underlining the versatility of this inoculum. the matched selenate-reducing bacterium tsa (ab018593) is closely related to the genus citrobacter that has been applied to reduce selenate from drainage waters containing 34 mg no 3 − l −1 and 338 µg se l −1 (zhang and frankenberger 2006) . surprisingly, it is a close relative to the clone from r1 resembling to the klebsiella species (b5-fj499346; fig. 5 ), although generally, dissmilatory selenate reducers are phylogenetically divers (stolz et al. 2006) . it is indicated that the selenate-reducing bacterium tsa (ab018593) is originating from the inoculum sludge, as it was matched in r2 and r3, only (fig. 5) . interestingly, a sulfurospirillum species also proliferated in the nonbioaugmented reactor. this study shows that the particle size of the bioprecipitates formed within the gel cubes is more than a factor 5 bigger (≤2µm, fig. 3c ) compared to previous studies (lenz et al. 2008a; oremland et al. 2004 ) using suspended cultures. as not subject to sheer forces, selenium precipitates formed by a membrane bound enzyme are not sloughed from the cell, and larger particle sizes can thus develop due to the cell immobilization in the gel. this effect might be used to select for larger bioprecipitates in certain applications (i.e., metal precipitation for recovery). precipitation within the gel separates part of the selenium from the water phase; thus, it does not leave the treatment plant with the effluent, preventing reoxidation in the environment (zhang et al. 2004 ). as the gel cubes did not float under the applied superficial upflow velocity, they can be easily recovered by settling for potential selenium re-use (lenz and lens 2009 ). however, technical processes for the re-use need to be developed. potentially, selenium could be recovered by heating of the gel cubes since polyacrylamide has a lower melting point compared to elemental selenium (102°c and 217°c, respectively). in contrast to previous studies (oremland et al. 2004 ), the precipitates consisted of selenium-sulfur mixtures (fig. 3d) , with sulfur either originating from sulfide used as reductant or sulfate present in the s. barnesii batch medium (see electronic supplementary material). as sulfate is present in many drainage waters (presser 1994 ) and thus biogenic sulfide can be formed, this will lower the selenium purity when considering re-use. the cementation of the cubes by inorganic precipitates from the feed medium (here calcium-phosphorous precipitates, fig. 3f and g) might limit substrate transport to the organisms (van langerak et al. 2000) during long time (>58 days) reactor operation. this study shows that regular uasb reactors can be applied to treat selenate-rich wastewaters under completely denitrifying conditions. however, in acute contamination situations, e.g., in case of spillages of ore processing waste (taggart et al. 2006) , the application of bioaugmented reactors can be advantageous due to the long start-up time required in regular uasb reactors (lenz et al. 2008a; lenz et al. 2008b ). however, it is necessary to pre-activate s. barnesii for such bioaugmentative applications to induce selenate reduction. this problem can potentially be avoided if selenium-respiring specialists that are unaffected by nitrate are bioaugmented to an already denitrifying sludge. the effluent se dis concentration of 39 µg se × l −1 achieved in r1 meets the current water quality criterion for salt waters (71µg se×l −1 ) set by the unites states environmental protection agency (usepa 2002). as filtration could further reduce se dis concentrations by removing selenium fines (between 450 and 100 nm particle size) from the r2 effluent, dissolved air flotation might be dispensable in practice, and slow sand filtration could be used as sole post-treatment of the effluent prior to emission [instead of the combination of both currently applied (green et al. 2003) ]. furthermore, the hydraulic retention times applied here are much shorter in contrast to the algalbacterial system (6 h versus 10-16 days; green et al. 2003) . it is important to note that no selenite was detected at any sample of r1, r2, or r3 (detection limit 24µg se×l −1 ) as selenite is more toxic to aquatic invertebrates and fish than selenate (hamilton 2004) . if formed, it is further reduced to elemental selenium by nitrite reductases or precipitated chemically with biogenic sulfide (hockin and gadd 2003) formed during reactor operation. if a classical uasb or hybrid system (uasb+immobilization gel cubes) is applied, an additional requirement of electron donor has to be taken into account to sustain the granular structure of the sludge (gonzalez-gil et al. 2001; santegoeds et al. 1999 ). here, a factor 2 excess lactate compared to nitrate was sufficient to maintain this structure. as most selenium containing streams are depleted in electron donor and its addition is the primary factor in the operating costs (zhang et al. 2008) , this excess should be minimized. as a result, utilization of immobilized cells alone might be more feasible, as less electron donor is consumed by non-selenium converting organisms. phylogeny of bacterial dgge sequences obtained from r2 and r3 biomass and an unfiltered effluent sample of r1 (obtained at the conclusion of the trial-day58) based on the kimura-2 model and the neighbor-joining method. bootstrap replicates (out of a total of 100 replicate samplings) that supported the branching order are shown at the relevant nodes. the scale bar represents five nucleotide substitutions per 100 sequence positions. genbank accession numbers are provided for sequenced dgge bands and reference sequences comparative bioavailability of selenium to aquatic organisms after biological treatment of agricultural drainage water bioaugmenting bioreactors for the continuous removal of 3-chloroaniline by a slow release approach is bioaugmentation a feasible strategy for pollutant removal and site remediation? temporal microbial diversity changes in solvent-degrading anaerobic granular sludge from low-temperature (15°c) wastewater treatment bioreactors advanced treatment technologies in the remediation of seleniferous drainage waters and sediments cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor selenium and nitrate removal from agricultural drainage using the aiwps (r) technology review of selenium toxicity in the aquatic food chain linked redox precipitation of sulfur and selenium under anaerobic conditions by sulfate-reducing bacterial biofilms a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences characteristics of and selection criteria for support materials for cell immobilization in wastewater treatment the essential toxin: the changing perception of selenium in environmental sciences selenium speciation in anaerobic granular sludge biological alkylation and colloid formation of selenium in methanogenic uasb reactors selenate removal in methanogenic and sulfate-reducing upflow anaerobic sludge bed reactors the ribosomal database project (rdp) reduction of selenium oxyanions in wastewater using two bacterial strains profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16 s rrna simultaneous reduction of nitrate and selenate by cell suspensions of selenium-respiring bacteria structural and spectral features of selenium nanospheres produced by se respiring bacteria geologic origin and pathways of selenium from the california coast ranges to the west-central san joaquin valley forecasting selenium discharges to the san francisco bay-delta estuary: ecological effects of a proposed san luis drain extension characterization of the reduction of selenate and tellurite by nitrate reductases the neighbor-joining method: a new method for reconstructing phylogenetic trees distribution of sulfate-reducing and methanogenic bacteria in anaerobic aggregates determined by microsensor and molecular analyses purification and characterization of the selenate reductase from thauera selenatis sulfurospirillum barnesii sp. nov. and sulfurospirillum arsenophilum sp. nov., new members of the sulfurospirillum clade of the ε proteobacteria arsenic and selenium in microbial metabolism phylogenetic analysis using parsimony (and other methods) after the aznalcollar mine spill: arsenic, zinc, selenium, lead and copper levels in the livers and bones of five waterfowl species the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools reduction of cr, mo, se and u by desulfovibrio desulfuricans immobilized in polyacrylamide gels national recommended water quality criteria high-rate sulfate reduction at high salinity (up to 90 ms.cm −1 ) in mesophilic uasb reactors impact of location of caco 3 precipitation on the development of intact anaerobic sludge microbial reduction of selenate and nitrate: common themes and variations thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor review of 15 years of research on ecotoxicology and remediation of land contaminated by agricultural drainage sediment rich in selenium removal of selenate in river and drainage waters by citrobacter braakii enhanced with zerovalent iron fate of colloidalparticulate elemental selenium in aquatic systems bacterial reduction of selenate to elemental selenium utilizing molasses as a carbon source acknowledgments the authors would like to thank ir. dai yue for her contribution to the laboratory reactor operations. this work was supported by the european union within the marie curie excellence team grant "novel biogeological engineering processes for heavy metal removal and recovery" (mextct-2003-509567).open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-011147-55whf8md authors: sun, hengchang; shang, mei; tang, zeli; jiang, hongye; dong, huimin; zhou, xinyi; lin, zhipeng; shi, cunbin; ren, pengli; zhao, lu; shi, mengchen; zhou, lina; pan, houjun; chang, ouqin; li, xuerong; huang, yan; yu, xinbing title: oral delivery of bacillus subtilis spores expressing clonorchis sinensis paramyosin protects grass carp from cercaria infection date: 2020-01-07 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-10316-0 sha: doc_id: 11147 cord_uid: 55whf8md clonorchis sinensis (c. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. effective strategies are still urgently expected to prevent against c. sinensis infection. in the present study, paramyosin of c. sinensis (cspmy) was stably and abundantly expressed on the surface of bacillus subtilis spores. the recombinant spores (b.s-cotc-cspmy) were incorporated in the basal pellets diet in three different dosages (1 × 10(5), 1 × 10(8), 1 × 10(11) cfu/g pellets) and orally administrated to grass carp (ctenopharyngodon idella). the immune responses and intestinal microbiota in the treated grass carp were investigated. results showed that specific anti-cspmy igm levels in sera, skin mucus, bile, and intestinal mucus, as well as mrna levels of igm and igz in the spleen and head kidney, were significantly increased in b.s-cotc-cspmy-10(11) group. besides, transcripts levels of il-8 and tnf-αin the spleen and head kidney were also significantly elevated than the control groups. moreover, mrna levels of tight junction proteins in the intestines of b.s-cotc-cspmy-10(11) group increased. potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 10(11) cfu/g b.s-cotc-cspmy spores administrated fishes could be detected compared with control group. the amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 10(11) cfu/g b.s-cotc-cspmy spores orally immunized group. our work demonstrated that b. subtilis spores presenting cspmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against c. sinensis infection for grass carp. electronic supplementary material: the online version of this article (10.1007/s00253-019-10316-0) contains supplementary material, which is available to authorized users. clonorchis sinensis (c. sinensis), an important fishborne zoonotic trematodes parasite, is prevalent in asian countries and regions including china, south korea, northern vietnam, and russia. adult worms of c. sinensis live in the intrahepatic bile duct of definitive host. in addition to human beings, various other kinds of mammals can be definitive hosts of c. sinensis, such as cat, dog, pig, rabbits, etc. as reported, the average prevalences of c. sinensis infection in dogs and cats were 20.5% and 41.8% in the pearl river delta region which is the most important endemic area in guangdong province (lin et al., 2011) . and nearly 35 million people are estimated to be infected with c. sinensis globally, of whom approximately 15 million are in china (lai et al. 2016; qian et al. 2016; tang et al. 2016b ) and bring a series of diseases like indigestion, biliary inflammation, bile duct obstruction, even liver cirrhosis, and hepatic carcinoma (tang et al. 2016b) . accumulating evidence demonstrated that there is an aetiological relation between clonorchiasis and cholangiocarcinoma in human beings (lun et al. 2005; machicado and marcos 2016; zheng et al. 2017 ). however, we still lack effective strategy to completely prevent the spread of c. sinensis at present (tang et al. 2016b) . human beings or other definitive hosts get infected by ingesting raw or undercooked fishes (the second intermediate hosts) containing live metacercaria (lun et al. 2005 ). on the one hand, in epidemic areas wild animals served as the definitive hosts (reservoir hosts) for c. sinensis (qian et al. 2016 ) which could be infectious source. for example, in southern china, a large number of dogs and cats roam freely in rural settings, and the presence of these animals in proximity with people may represent a risk of parasitic zoonoses including c. sinensis (fang et al. 2015; nguyen et al. 2018 ). on the other hand, eating raw fish has been deeply rooted in culture of the area. in previous, most vaccine trials focused on the definitive host of c. sinensis instead of the intermediate hosts including freshwater fishes or snails (the first intermediate hosts). protein-based or nucleic acid-based vaccine trials have been conducted on the rat model, but none of the vaccine candidates brought a protective effection (worm reduction rate) of more than 70% (qian et al. 2016; tang et al. 2016b ). freshwater fishes (e.g., ctenopharyngodon idellus, carassius auratus, and hypophthalmichthys nobilis) serve as the second intermediate host for c. sinensis. hence, we speculate that cutting off the life cycle of c. sinensis by preventing the cercaria invasion or metacercariae formation in freshwater fish might be an efficacious tactic to control the prevalence of c. sinensis. vaccine has been the most effective method for combating infectious disease in aquaculture industry (gudding and van muiswinkel 2013; plant and lapatra 2011) . compared with other immunization routes (injection and immersion route), oral vaccine is a preferable route as it is needle-free, no size limitation, lower cost, and more convenient for farmer operation (plant and lapatra 2011) . however, oral immunization suffers from antigen degradation in the gastrointestinal tract of fish, which will affect the protective effect (quentel and vigneulle 1997) . a considerable amount of investigations have proved that spores of bacillus subtilis (b. subtilis) were a potent antigen delivery platform for oral vaccine rosales-mendoza and angulo 2015; tavares batista et al. 2014) . because b. subtilis spores can survive extreme environment in the gastrointestinal tract, thus protect the antigens from digestion and degradation (duc le et al. 2003) . besides, b. subtilis were widely employed as probiotic additives as it enhances the growth performance, digestive enzyme activities, immune responses, and disease resistance of fishes or shrimps (liu et al. 2017; sanchez-ortiz et al. 2016; truong thy et al. 2017; wang et al. 2010) . b. subtilis spores were widely investigated as a delivery vehicle for the oral vaccine in aquaculture industry (fu et al. 2010; valdez et al. 2014) . in our previous work, an oral delivery system based on b. subtilis spore has been successfully established and confirmed to be valid and feasible tang et al. 2017; zhou et al. 2008) . enolase and cysteine protease of c. sinensis (cseno and cscp) were expressed on the surface of b. subtilis spore, and the recombinant spores elicited both humoral and mucosal immune response in grass carp by oral immunization tang et al. 2017) . but the protect effect against c.sinensis and the safety of spores need further study. paramyosin (pmy), an invertebrate muscle-associated multifunctional protein, has emerged as a promising vaccine candidate for various kinds of parasites (e.g., schistosoma mansoni, schistosoma japonicum, taenia solium, fasciola gigantica, etc.) (abou-elhakam et al. 2013; jiz et al. 2015; vazquez-talavera et al. 2001) . paramyosin of c. sinensis (cspmy, accession number: jq041818.1) was found to be highly expressed at the stage of adult worm, metacercariae, and cercaria. both prokaryotic expressed protein and dna vaccine of cspmy brought encouraging protect effect in rat models. furthermore, cspmy was confirmed to be an important component of cyst wall of metacercariae, which suggested us that cspmy may play a vital role in metacercariae formation in freshwater fishes ). in the present study, we aimed to explore whether oral administration with b. subtilis spores expressing cspmy on the surface would be an effective and safe measure to protect grass carp from c. sinensis infection. cspmy were fusion expressed on the surface of b. subtilis spores with cotc, a coat proteins of b. subtilis spores, and the immune response and protect effect in grass carp elicited by oral administration with the recombinant spores were evaluated. besides, its influence on intestines and the intestinal microbiota of immunized grass carp were also investigated by using qrt-pcr and miseq high-throughput sequencing, respectively. fishes and recombinant b. subtilis spores healthy grass carp weighing 20-25 g were acquired from seedling production base of pearl river fisheries institute (guangzhou, china) and kept in the laboratory for acclimation for 2 weeks. subsequently, fishes were divided into several tanks with the same volume of water (35 fishes per tank) and fed daily. before experiments, fishes were randomly sampled for metacercaria detection according to the methods described previously (liang et al. 2009 ) to confirm negative infection. b. subtilis spore fusion expressing cotc-cspmy (b.s-cotc-cspmy) and b. subtilis spore expressing cotc (b.s-cotc) as the control were obtained by our previous study and preserved in our lab (sun et al. 2018 ). extraction of coat protein of spores b. subtilis wb600 strains with b.s-cotc-cspmy or b.s-cotc was cultured in difco sporulation medium (dsm, bd, franklin lakes, usa) for 24 h as described. spores were harvested and washed with 1 m nacl, 1 m kcl and distilled water in turn (nicholson wl 1990) . finally, spores were resuspended in distilled water and treated in 68°c for 1 h to kill residue vegetative cell. they were counted and stored in − 80°c prior to use. to extract the coat proteins of spores, spores were resuspended with sodium dodecyl sulfate (sds)-dithiothreitol (dtt) extraction buffer (0.5% sds, 0.1 m dtt, 0.1 m nacl) and incubated at 37°c for 2 h (tang et al. 2016a) . followed by six times wash with 1 m tris-hcl buffer (ph 8.0), the spores were suspended in 5 ml broken buffer (50 mm tris-hcl, 0.5 mm edta, 1 mm pmsf) and ultrasonicated for 5 min. after centrifugation, the coat proteins were collected from the sediment, and the supernatant was preserved for further analysis as well (tang et al. 2016a; zhou et al. 2008) . b.s-cotc-cspmy spores were analyzed by 12% sds-page. the corresponding expression band of cotc-cspmy in the polyacrylamide gel was digested with trypsin as described before (katayama et al. 2001) . the peptides were analyzed through liquid chromatography coupled with tandem mass spectrometry (lc-ms/ms) with an ltq orbitrap velos pro mass spectrometer (thermo finnigan, usa). mascot v2.3 search engine (matrix science, london, uk) was applied for protein identification using the following search parameters: clonorchis sinensis database; two missed cleavage site; fixed modifications of carbamidomethyl (c); partial modifications of acetyl (protein n-term), deamidated (nq), deoxidation (w), oxidation (m); and ± 30 ppm for precursor ion tolerance and ± 0.15 da for fragment ion tolerance. the basal diet was a commercial pellet (taifeng, foshan, china, with the chemical composition of crude protein ≥ 30.0%, crude fat ≥ 3.0%, ash ≤ 13.0%, crude cellulose ≤ 12.0%, lysine ≥ 1.3%, and total phosphorus ≥ 0.7%). five experimental diets were prepared in accordance with the methods described before tang et al. 2017) . naïve group was treated with basal feed without spores. the control group (b.s-cotc-10 8 group) was administrated with basal feed plus b.s-cotc spores (1 × 10 8 cfu/g). experimental groups were managed with 1 × 10 5 cfu/g, 1 × 10 8 cfu/g, or 1 × 10 11 cfu/g of b.s-cotc-cspmy spores. to avoid dispersion of the spores in water, spores and basal feed were coated with an equal volume of cod liver oil . the diets were dried and stored at − 20°c until use. for oral immunization, fishes in each group were hand-fed with 2% of their initial body weight twice per day (at 09:00 am and 17:00 pm) for 6 weeks. thereafter, fishes were fed with basal diet till the end of the experiment. five fishes were randomly sampled from each group at week 2, 4, and 6 after the beginning of the immunization. grass carp was euthanized with overdose of eugenol mixture, and then skin mucus, blood, gallbladder, intestinal mucus, head kidney, and spleen were collected as described method (guo et al. 2016) . briefly, skin mucus was gently scraped with a glass slide, then diluted with 0.5 ml sterile pbs, centrifuge at 4°c, 4500 rpm for 20 min, and stored in −80°c ). blood was collected from the caudal vein using a 1 ml injector, clotted in the room temperature for 2 h, and then sera were separated by centrifugation (4°c, 4000 rpm for 15 min) and stored in − 20°c until use . the intestine was aseptically isolated, lavaged with 0.5 ml sterile pbs for several times, and centrifugated (4°c, 4500 rpm for 20 min). the supernatant of the lavage fluid was stored at − 20°c. the spleen and head kidney of each fish was dissected and preserved in sample protector (takara bio, otsu, japan) in − 80°c. about 8 weeks after the beginning of administration, another three fishes in each group were sampled and euthanized, and the whole intestines of the three fishes were aseptically excised for gut microbiota analysis. indirect enzyme-linked immunosorbent assay (elisa) was employed to analyze the specific antibody levels against cspmy in samples including skin mucus, sera, bile, and intestinal mucus. in brief, the 96-well microtiter plates were coated with 100 ml per well of 5 μg/ml rcspmy carbonatebicarbonate buffer (0.05 m, ph 9.6) in 4°c overnight. after washing with pbst three times, the plates were blocked with blocking buffer containing 5% skimmed milk for 2 h at 37°c. meanwhile, sera, bile, skin mucus, and intestinal mucus was diluted with 1% bsa in pbst at a dilution of 1:100, 1:100, 1:20, 1:20, respectively. then 0.1 ml of diluted samples were added to each well as primary antibody and incubated for 2 h at 37°c. after three times washing, 0.1 ml of hrp-conjugated rabbit anti-grass carp igm (diluted at 1:7000) was added and incubated at 37°c for 1 h. the plates were washed for 3 times again, 0.1 ml of tetramethylbenzidine (tmb, bd, usa) was added and reacted at rt for 10 min. finally, the reaction was terminated by adding 50 μl of 2 m h 2 so 4 , and the optical density value at 450 nm was detected by a microplate reader. mrna levels of immune-related molecules and tight junction proteins by quantitative real-time polymerase chain reaction (qrt-pcr) about 6 weeks after the beginning of oral administration, total rna was extracted from the head kidney, spleen, foregut, midgut, or hindgut tissues of grass carp with trizol reagent (transgen biotech, beijing, china) and was reverse transcribed into the first strand of cdna by using an all-in-one first-strand cdna synthesis supermix kit (transgen biotech, beijing, china). mrna levels of immunoglobulin m (igm), immunoglobulin z (igz), tumor necrosis factor α (tnf-α), and interleukin 8(il-8) in the head kidney and spleen were analyzed by qrt-pcr. additionally, transcription levels of gene encoding tight junction proteins including zo-1, occludin, claudin b, and claudin c were also detected. βactin was used as an internal reference gene . the specific primers were designed according to the published grass carp sequences and were listed in table s1 . the qrt-pcr procedure was carried out by the process described ). data were analyzed by the 2 -δδct method with cfx manager software. the whole intestines of the three fishes were aseptically excised and opened and rinsed with sterilized pbs to remove the contents. intestines from three fishes in the same group were collected into one sterile centrifuge tube. the intestinal samples were homogenized with sterilized pbs. the genomic dna was extracted using omega d3350 bacterial dna kit (omega, usa) according to manufacturer's protocols. the v3-v4 regions of 16s ribosomal dna of bacteria were amplified, purified, and pooled in equimolar and paired-end sequenced (2 × 250 bp) on an illumina miseq platform (oe biotech, shanghai, china). data was demultiplexed, quality filtered, and analyzed with qiime(v1.8.0) and uchime (v4.2) (caporaso et al. 2010; edgar et al. 2011) . operational taxonomic units (otus) were generated using vsearch (v2.4.2) software with 97% similarity cutoff. all representative reads were annotated and blasted against silva database (v123) using rdp classifier (v2.2) with the confidence threshold of 70% (hao et al. 2017a ). bias-corrected chao1 richness estimator (chao 1) was applied to evaluate the community richness. shannon-wiener index (shannon) and simpson's diversity index (simpson) was used for diversity evaluation for each sample. the structures of the microbial community in different samples were compared based on the column diagram, heat map analysis, and principal-component analysis (pca) (hao et al. 2017a ). the bacterial abundance was analyzed among the groups. all the illumina sequencing data has been deposited in the ncbi sequence read archive database (http://www.ncbi.nlm.nih.gov/sra/), and the accession n u m b e r s w e r e s r r 1 0 2 4 4 3 8 5 / s r r 1 0 2 4 4 3 8 4 / srr10244383/srr10244382/srr10244381. parafossarulus striatulus, the first intermediate host of c. sinensis, were captured from yangshan county, guangdong province, china, and were elaborately cultivated in our laboratory. after 2 weeks acclimation, the parafossarulus striatulus were fed with c. sinensis eggs. ninety days later, snails were checked every day for cercariae release (liang et al. 2009 ). water with living cercariae was collected every day and was equally divided into each tank to infect fishes in the naïve group (n = 5), b.s-cotc-10 8 group (n = 5), and b.s-cotc-cspmy-10 11 group (n = 5). challenge infection lasted for 7 days. four weeks post the challenge infection, all fishes were sacrificed. the flesh was weighted, cut into pieces, and separately digested with artificial gastric juice for c.sinensis metacercaria detection. the number of metacercaria in per gram flesh of fish was calculated. data in experiments were expressed as the mean ± sd values. student's t test was applied to analyze statistical differences in mrna levels and number of metacercaria in per gram flesh among different groups using graphpad prism 5 software (version 5.0 for windows). one-way analysis of variance (anova) with tukey tests were used to analyze statistical differences of antibody levels with graphpad prism 5 software (version 5.0 for windows). the difference was considered as statistically significant if the p value < 0.05. sds-page and western blotting by using anti-cspmy antibody showed that cotc-cspmy expressed in coat proteins extracted from the recombinant b. subtilis spores (fig. 1a, b) . flow cytometry showed that 58.4% of b.s-cotc-cspmy spores exhibited a strong fluorescence intensity ranging from 10 4 to 10 6 by anti-cspmy serum as a primary antibody (fig. 1c) . lc-ms/ms confirmed the protein band with 100 kda as cspmy ( fig. 1d and table s2 ). specific igm levels in sera, skin mucus, intestinal mucus, and bile compared with naïve groups and b.s-cotc groups, specific anti-cspmy igm levels in sera, skin mucus, intestinal mucus, and bile samples from fishes of b.s-cotc-cspmy groups raised since week 2 and went on rising till week 6 ( fig. 2) . in the skin mucus, igm level significantly elevated from week 4 ( fig. 2b) , which raised later than those in sera, intestinal mucus, and bile samples ( fig. 2a, c, d) . compare with the 10 5 dosage group, 10 8 and 10 11 dosage groups elicited higher igm level. the highest one was 10 11 dosage group (fig. 2) . mrna levels of igm and igz in the head kidney and spleen compared with naïve groups, the mrna levels of igm and igz in middle dosage (1 × 10 8 cfu/g b.s-cotc-cspmy spores) and high dosage (1 × 10 11 cfu/g b.s-cotc -cspmy spores) were significantly increased both in the head kidney and spleen. the mrna levels of igm and igz in 1 × 10 11 cfu/g groups were the highest. there was no significant difference in mrna levels of igm and igz in the head kidney and spleen between naïve group and b.s-cotc group. compared with naïve group, there was no statistical increase of mrna levels of igm and igz in 1 × 10 5 cfu/g group except igz mrna level in the head kidney (fig. 3 ). in the head kidney, tnf-α mrna level of 1 × 10 11 cfu/g b.s-cotc-cspmy group were significantly higher than those in naïve group and b.s-cotc group (fig. s1a ), while no significant differences were detected between those in 1 × 10 5 and 1 × 10 8 cfu/g b.s-cotc-cspmy groups. il-8 mrna level was significantly raised compared with both naïve group and b.s-cotc group. in spleen, mrna levels of tnf-αand the paramyosin from c. sinensis was identified in coat proteins il-8 significantly increased in 1 × 10 8 and 1 × 10 11 cfu/g b.s-cotc-cspmy groups compared with those in naïve group (fig. s1b ). no differences were detected between b.s-cotc and naïve group (fig. s1 ). to investigate the influences of oral administration of spores on the integrality of intestines mucosa, transcriptional levels of genes encoding tight junction proteins (zo-1, occludin, claudin b, claudin c) in foregut, midgut, and hindgut of grass carp were analyzed. in the foregut, transcriptional levels of zo-1, occludin, claudin b, and claudin c were the highest in fishes fed with 1 × 10 11 cfu/g b.s-cotc-cspmy spores, which were significantly increased compared with those in fishes from naïve or b.s-cotc groups (fig. s2a) . the zo-1 mrna level in the midgut and hindgut were not dominantly affected by spores administration (fig. s2b-a, c-a) . transcriptional levels of occludin, claudin b, claudin c in midgut, and hindgut of grass carp from 1 × 10 11 b.s-cotc-cspmy spores group were obviously elevated compared with those of other groups (fig. s2b, c) . in total, 963 otus were generated from the samples through illumina miseq sequencing, and these otus belong to 11 different phyla. good's nonparametric coverage estimator (good's coverage) of each sample tend to approach 1 ( fig. 4aa) . chao 1 index was used to estimate microbial community richness of the intestinal microbiota. the chao 1 value in 1 × 10 11 cfu/g b.s-cotc-cspmy spores (bh) group was the highest compared with those of other groups ( fig. 4a-b) . shannon index and simpson index were calculated to evaluate microbial community diversity. shannon and simpson index value of bh group was the highest among the groups. diversity index of b.s-cotc spores fed group (bc), 1 × 10 5 / 10 8 / 10 11 cfu/g b.s-cotc-cspmy spores treated groups (bl, bm, bh) were higher than that of control group (nc) (fig. 4a-c, d) . the shared and unique outs among the samples were investigated and showed in the venn diagram. a total of 84 otus were shared by all the 5 groups, and 45 unique outs were detected in bh group, which was the largest number among groups (fig. 4b) . principal components analysis (pca) with weighted unifrac method was applied to analyze the relationships between bacterial communities from different groups. the score plot of pca revealed that microbial communities from bc, bl, bm, and nc groups were gathered at left-hand side of the plot along the second principal component axis (pc2), which accounts for 72.1% of the total variations. however, bh group was on right side of the graph along pc2. bl group was separated from the other groups along pc1, which represented 14.08% of the total variations. in general, the two axes (pc1 and pc2) explained 86.18% (fig. 4c ). in addition, the b.s-cotc-10 8 : basal diet plus 1 × 10 8 cfu/g b.s-cotc spores. b.s-cotc-cspmy-10 5 , 10 8 , 10 11 , basal diet plus 10 5 , 10 8 , 10 11 cfu/g b.s-cotc-cspmy spores, respectively. data were represented as mean ± sd. statistical significance was analyzed by the dunnett's test. *p < 0.05, **p < 0.01, ***p < 0.001 differences among the groups were also observed in the heatmap, which showed the bacteria ranking top 30 in the abundance. (fig. 4d) . proteobacteria, fusobacteria, firmicutes, and spirochaetae were main bacteria phyla in all grass carp, accounting for more than 99% of bacteria. firmicutes were the most predominant phylum in bh group (25.38%), followed by fusobacteria (8.2%). in nc, bc, and bm groups, proteobacteria had the highest abundance accounted for 23.74%, 58.55%, and 51.15%, respectively (table 1) . the abundance of potential pathogenetic bacteria such as pseudomonas and flavobacterium were detected in samples. the amount of sequences related to pseudomonas in bh group decreased compared with that in nc group. flavobacterium with lower abundance was found in nc and bl groups but not in bh, bc, and bm groups. (table 2) . candidate probiotics including lactobacillus, streptococcus, and micrococcus were also detected and analyzed. higherabundance lactobacillus and streptococcus genus were found in bh group compared with nc group. micrococcus could not be detected in all samples (table 2 ). in addition, sequences related to bacteria associated with digestion were also investigated. the abundance of rikenellaceae, ruminococcaceae, and lachnospiraceae (at family level) varied in different groups, but they were dramatically increased in bh group compared with other groups ( table 3 ). the abundances of odoribacter, desulfovibrio, and alistipes were higher in bh group than those in nc, bl, and bm groups. after the challenge infection with living cercaria (fig. 5a) , c. sinensis metacercaria (fig. 5b ) was found in all treated fishes. the average amount of metacercaria per gram fish flesh in the naïve group, b.s-cotc-10 8 group, and b.s-cotc-cspmy-10 11 group was 13.2, 12.4 and 7.2, respectively (fig. 5c ). in the current study, we orally administrated grass carp with 1 × 10 5 , 1 × 10 8 or 1 × 10 11 cfu/g b. subtilis spores surface displaying cspmy. the results showed that specific anti-cspmy igm levels in sera, skin mucus, intestinal mucus, and bile samples from fishes in b.s-cotc-cspmy spores orally treated groups dominantly increased. in 1 × 10 11 cfu/g b.s-cotc-cspmy spores administrated fishes, mrna levels of fig. 3 the relative mrna levels of igm and igz in the head kidney and spleen. the mrna levels of igm and igz were analyzed by qrt-pcr at 6 weeks after the beginning of the immunization. (a) igm mrna level in the head kidney; (b) igz mrna level in the head kidney; (c) igm mrna level in the spleen; (d) igz mrna level in the spleen. naïve, basal diet group; b.s-cotc-10 8 : basal diet plus 1 × 10 8 cfu/g b.s-cotc spores. b.s-cotc-cspmy-10 5 , 10 8 , 10 11 , basal diet plus 10 5 , 10 8 , 10 11 cfu/g b.s-cotc-cspmy spores, respectively. *p < 0.05, **p < 0.01 (compared with naïve group); #p < 0.05, ## p < 0.01 (compared with b.s-cotc-10 8 group) igm, igz, tnf-α, and il-8 significantly elevated in the head kidney and spleen, and transciptional levels of tight-junction proteins in foregut, midgut, and hindgut of grass carp obviously increased too. gut microbiome indicated that potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 10 11 cfu/g b.s-cotc-cspmy spores administrated fishes were detected. the amount of metacercaria in per gram fish flesh were statistically decreased in 1 × 10 11 cfu/g b.s-cotc-cspmy spores orally immunized group. in the past years, the prevention and management of the c. sinensis mainly relied on an integrated control strategy including chemotherapy of infected patients, management or numbers represent the relative abundance of the predominant bacterial phyla. nc, fishes were fed with basal diet; bc, fishes were fed with pellets covered with b.s-cotc spores (1 × 10 8 cfu g −1 pellets); bl, bm, bh, fishes were fed with pellets covered with dosages of 1 × 10 5 , 1 × 10 8 , 1 × 10 11 cfu g −1 pellets of b.s-cotc-cspmy spores, respectively sterilization of feces, implementation of education campaigns, etc. lun et al. 2005; qian et al. 2016 ). this strategy helped to reduce the infection rate of c. sinensis in human beings to a certain degree (qian et al. 2016 ), but surveys showed that, in some epidemic areas, the prevalence of c.sinensis has been increasing over the years (lun et al. 2005 ). hence, vaccine or more effective drugs are urgently expected to control the spread of c. sinensis. a lot of research works had explored vaccine against c.sinensis in recent years, but almost of them were focused on vaccine for the final host (tang et al. 2016b ). to our knowledge, eating raw or undercooked fish flesh harboring living metacercaria is the only route for human being or animals to get infected with c. sinensis. in view of this status, we speculated that cutting off the life cycle of c. sinensis by interfering the metacercariae formation in freshwater fish might be a potential strategy. in our previous work, oral vaccines based on b. subtilis spore expressing enolase or cysteine protease of c. sinensis has already been tested on grass carp and proved to be able to induce both system and local mucosal immune response of fish tang et al. 2017) . but immune protect effect elicited from vaccine candidates besides the two molecules still needs further evaluation and confirmation . cspmy, as a multifunctional molecular and important component of cyst wall of c. sinensis metacercaria, has been well characterized as a vaccine candidate ). in the current study, cspmy was displayed on the surface of b. subtilis spores by using a coat protein (cotc) as an anchor. sds-page, western blotting, flow cytometry, and lc-ms/ms verified the successful expression of cspmy (fig. 1) . recombinant b. subtilis spores are firstly took in by the m cells in intestinal mucosa and transported into peyer's patches. antigens presented on the surface of spores interact with mucosa-associated lymphoid tissues (malts) and other systemic lymphoid tissues, resulting in a series of immune responses including secretion of immune globulins (rosales-mendoza and angulo 2015; tang et al. 2016a; tavares batista et al. 2014) . in bony fishes, as there are a lot of lymphoid cells, macrophages, eosinophilic, neutrophilic granulocytes, and other secretory cells in skin, it is also a main tissue-producing mucosal immune response besides intestinal mucosa (salinas 2015) . antigen-specific immune globulins (e.g., igm and igz) were secreted from intestinal mucus and skin mucus when they were stimulated by heterogeneous antigens so that they serve as the first line of immunological barriers against pathogenic invasion (zhu et al. 2013 ). well-studied igm is the main kind of immune globulins in grass carp. igz, analogous to mammalian iga, also plays a specialized role in mucosal immune responses of fish (xu et al. 2013 ). in the present study, the specific igm antibody levels in serum, bile, intestinal mucus, and skin mucus of grass carp orally administrated with different dosages of spores (b.s-cotc-cspmy) were significantly increased with dosedependent from the 2nd week after the beginning of the immunization till to 6 weeks (fig. 2) . our results were coincident with the former reports about immune response in grass carp induced by oral immunization with spore expressing enolase or cysteine protease of c. sinensis tang et al. 2017) . moreover, mrna levels of igm and igz were up-regulated in the head kidney and spleen of fishes from b.s-cotc-cspmy spores orally treated groups (fig. 3) . the results verified that cspmy on the spore surface could be recognized by immune system in intestinal mucosa and obviously elicit strong systemic and local mucosal immune response in grass carp by oral administration, especially treated with high dosage (b.s-cotc-cspmy-10 11 ). the numbers represent the total abundance of bacteria genus presented in the community. nc, fishes were fed with basal diet; bc, fishes were fed with pellets covered with b.s-cotc spores (1 × 10 8 cfu g −1 pellets); bl, bm, bh, fishes were fed with pellets covered with dosages of 1 × 10 5 , 1 × 10 8 , 1 × 10 11 cfu g −1 pellets b.s-cotc-cspmy spores, respectively # at family level *at genus level the numbers represent the total abundance of bacteria genus presented in the community. nc, fishes were fed with basal diet; bc, fishes were fed with pellets covered with b.s-cotc spores (1 × 10 8 cfu g −1 pellets); bl, bm, bh, fishes were fed with pellets covered with dosages of 1 × 10 5 , 1 × 10 8 , 1 × 10 11 cfu g −1 pellets b.s-cotc-cspmy spores, respectively * represent the potential pathogenetic bacteria # the candidate probiotics the specific igm levels in the intestinal mucus and bile of grass carp increased more early than that in sera or skin mucus in b.s-cotc-cspmy groups (fig. 2) . it might due to that orally delivered antigen (cspmy) interacted with the local immune system on the intestinal mucosa first and later triggered the systemic immune reaction. it has been documented that immune status of grass carp was closely related to expression of cytokines (secombes et al. 2001 ). il-8 was considered to be one of the most important proinflammatory cytokines in grass carp (wang et al. 2013) . il-8 expresses in immune-related tissues in grass carp such as the head kidney, spleen, gill, and so on. il-8 level would increase when grass carp was stimulated by heterologous antigens or pathogens resulting in attraction of other immunerelated cells for fighting against the pathogens (wang et al. 2013) . tnf-α was also proved to be a crucial cytokine involved in immune regulation and mediates the inflammatory responses in grass carp zhu et al. 2013 ). in the current study, mrna levels of tnf-α and il-8 in the head kidney and spleen in b.s-cotc-cspmy-10 8 and 10 11 groups were significantly up-regulated at week 6 ( fig. s1 ). it demonstrated that oral administration of the recombinant spores activated the innate immunity and might invoke specific adaptive immune responses . additionally, the tnf-αlevel had no significant difference in b.s-cotc-cspmy 1 × 10 5 and 1 × 10 8 group in the head kidney. the possible reason might be that the spore dosages (1 × 10 5 and 1 × 10 8 cfu/g) were not enough to induce tnf-αsecretion in the head kidney because a certain amount of the spores might be excreted from the intestine with the intestinal movement (leser et al. 2008) . studies showed that the structural integrity of intestinal mucosa was crucial to vaccinated fishes . tight junction proteins play vital roles in tight junction among epithelial cells of intestine which maintains intestinal integrity resulting in the foundation of mechanical barrier (chen et al. 2015) . evidences indicated that expressions of tight junction proteins such as zo-1, occludin, claudin b, or claudin c would be down-regulated when intestinal mucosa was damaged by inflammation (chen et al. 2015; feng et al. 2015; gong et al. 2016) . our results showed that oral administration with 10 8 or 10 11 cfu/g − b. subtilis spores up-regulated mrna levels of occludin, claudin b, and claudin c in foregut, midgut, and hindgut. mrna levels of zo-1 increased in the foregut (fig. s2) . it was previously mentioned that b. subtilis could promote epithelial tight junctions of mice with inflammatory bowel disease (gong et al. 2016) , indicating that b. subtilis spores were beneficial to maintenance of structural integrity of intestinal mucosa in grass carp. the gastrointestinal microflora is a complex ecosystem harboring a variety of bacterial communities and plays an imperative role in immune regulation and nutrition consumption of the host (han et al. 2010; hao et al. 2017a; hao et al. 2017b) . variation in the diversity and abundance of bacteria in intestine seriously impairs the immune development and function (nayak 2010) . it has been verified that probiotic additive (e.g., b. subtilis) enhance certain innate immune response, thus improve disease resistance of the host (hao et al. 2017a; li et al. 2016; liu et al. 2015) . in addition, probiotic supplement in the diets improves growth performance by positively improving the composition of intestinal microbial community (hao et al. 2017a) . we analyzed intestinal microbiota by using 16s rrna sequencing. our results indicated that proteobacteria, firmicutes, and fusobacteria were the dominant phyla in each group (table 1 ), suggesting that dietary supplement of b. subtilis spores did not affect the primary phyla constitution of grass carp. compared with naïve group, both the diversity and richness indices of bacteria in intestines of spores administrated grass carp were elevated, especially in b.s-cotc-cspmy-10 11 group. the results of pca, venn, and heatmap also verified that the intestinal microbiota in b.s-cotc-cspmy-10 11 group was dramatically different from the other groups (fig. 4) . previous studies reported that b. subtilis ch9 and b. subtilis cgmcc 1.1086 strains could increase probiotics but decrease pathogenetic bacteria in intestinal of grass carp or broilers (hao et al. 2017a; li et al. 2016) . streptococcus, lactobacillus, and micrococcus have been widely used as probiotics in aquaculture and brought positive effects on the host including improvement of the immune status and inhibiting pathogen infection in the intestines (balcazar et al. 2006; hai 2015) . in our present study, the abundance of streptococcus and lactobacillus in high-dosage spores-treated group was dramatically higher than those in other groups (table 2) , while lactobacillus was not detected in intestine of b. subtilis treated fish (hao et al. 2017a) . considering that culture condition and feed may affect intestinal microbiota of fish (han et al. 2010; wu et al. 2012) , it might due to the different culture condition and diet in different experiments. some strains from flavobacterium and pseudomonas genera were considered to be potential pathogens to grass carp, as they might induce columnaris disease, white head-mouth disease, and red skin disease (hao et al. 2017a) . lower abundance of flavobacterium was found in naïve group but not in b. subtilis treated groups. dramatically lower abundance of pseudomonas in b.s-cotc-cspmy-10 11 group was detected compared with naïve group ( table 2) . the results demonstrated that oral administration with probiotics might reduce potential pathogenic bacteria in grass carp intestine (hao et al. 2017a; liu et al. 2015) . a possible explanation might be that probiotics generate antibiotics, bacteriocins, hydrogen peroxide, and lysozyme, etc., thus inhibit the adherence or proliferation of pathogens in intestinal mucosa (hai 2015; sugita et al. 1998 ). in addition, rikenellaceae, ruminococcaceae, and lachnospiraceae were dramatically increased in b.s-cotc-cspmy-10 11 group. at genus level, higher abundance of alistipes, odoribacter, and desulfovibrio in the high-dosage group were presented (table 3) . rikenellaceae were considered as intestine beneficial bacteria, as alistipes genus belong to the family can produce fibrinolysin, digest gelatin, and ferment carbohydrate resulting in enhancement of digestion in fishes . odoribacter can produce short-chain fatty acids (scfas) such as acetic acid, succinic acid, or butyric acid, which are beneficial to both microbial and epithelial cell growth of host (meehan and beiko 2014) . desulfovibrio has also been proved to be beneficial to the microbial community and able to improve energy recovery from food . lachnospiraceae are a butyrate-producing superfamily, which can produce an enzyme to break down a wide variety of complex carbohydrates found in plants (hao et al. 2017b) . hence, higher abundances of these bacteria found in the intestines would probably be helpful to digestion function of grass carp and promote their growth performance. compared with naïve group, abundances of bacteria mentioned above were not obviously changed in bc, bl, and bm group (b.s-cotc-10 8 , b.s-cotc-cspmy-10 5 /10 8 groups) (tables 1, 2, 3) . it might because that lower dosage of b. subtilis spores accompanied by less colonized b. subtilis spores in intestine of grass carp (hao et al. 2017a) . after oral immunization, the fishes were infected with c. sinensis cercaria for a week. compared with naïve group, metacercaria intensities in b.s-cotc-cspmy-10 11 spore treated grass carp were significantly decreased (fig. 5c ). we conjectured that immune responses induced by the recombinant spores (b.s-cotc-cspmy) played roles in protecting against adherence or invasion of cercaria to grass carp. it needed further study to illuminate the involved mechanisms. when grass carp was orally administrated with b.s-cotc-cscp spore (10 7 dosages), no metacercariae were observed . we speculated that might be due to two reasons: firstly, the function and immunogenicity of cspmy were different from those of cscp (tang et al. 2016a; wang et al. 2012) resulting in different protect effect. secondly, the methods used for metacercariae detection were different. in the former study, metacercariae were checked by direct compression method by randomly picking fish flesh from 5 different positions in one fish , which might lead to missed detection when the infection intensity was low. while in our present study, we checked by digestion method with high detection rate. that is, the fishes were totally digested with artificial gastric juice, so that every metacercariae could be detected. in conclusion, oral administration with b.s-cotc-cspmy spores could induced both systemic and local mucosal immune responses without bad effects on intestinal structural integrity and elicited promising protective effect in grass carp. additionally, oral treatment of the spores in grass carp could reduce the abundance of potentially pathogenic bacteria (e.g., flavobacterium) and enhance the abundance of probiotics (e.g., streptococcus, lactobacillus) or bacteria associated with digestion. our work suggested that b. subtilis spore presenting cspmy on the surface is a promising effect, safe, and needlefree oral vaccine candidate for prevention of c. sinensis infection in grass carp. our study established the cornerstone for the prevention of c. sinensis infection in both human and mammalian reservoir hosts by using fish vaccine 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ctenopharyngodon idellus composition, diversity, and origin of the bacterial community in grass carp intestine teleost skin, an ancient mucosal surface that elicits gut-like immune responses functional characterization of tnf-alpha in grass carp head kidney leukocytes: induction and involvement in the regulation of nf-kappab signaling liver fluke infection and cholangiocarcinoma: a review oral administration of a bacillus subtilis sporebased vaccine expressing clonorchis sinensis tegumental protein 22.3 kda confers protection against clonorchis sinensis advances in research of fish immune-relevant genes: a comparative overview of innate and adaptive immunity in teleosts publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-302503-7s9f8wje authors: fu, yuguang; li, baoyu; liu, guangliang title: rapid and efficient detection methods of pathogenic swine enteric coronaviruses date: 2020-05-19 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10645-5 sha: doc_id: 302503 cord_uid: 7s9f8wje abstract: porcine enteric coronaviruses (covs) cause highly contagious enteric diarrhea in suckling piglets. these cov infections are characterized by clinical signs of vomiting, watery diarrhea, dehydration, and high morbidity and mortality, resulting in significant economic losses and tremendous threats to the pig farming industry worldwide. because the clinical manifestations of pigs infected by different covs are similar, it is difficult to differentiate between the specific pathogens. effective high-throughput detection methods are powerful tools used in the prevention and control of diseases. the immune system of piglets is not well developed, so serological methods to detect antibodies against these viruses are not suitable for rapid and early detection. this paper reviews various pcr-based methods used for the rapid and efficient detection of these pathogenic covs in swine intestines. key points: 1. swine enteric coronaviruses (covs) emerged and reemerged in past years. 2. enteric covs infect pigs at all ages with high mortality rate in suckling pigs. 3. rapid and efficient detection methods are needed and critical for diagnosis. the coronaviruses (covs) critically threaten human and animal health because infection with them results in respiratory or enteric tract diseases (woo et al. 2012) . for instance, pneumonia occurs in humans infected with the new cov that emerged in china in the middle of dec. 2019; the virus has subsequently spread to many countries worldwide where it threatens the health of humans, resulting in tremendous economic losses. covs are enveloped, positive-sense, singlestranded rna viruses that possess a genome ranging from 25.4 to 31.7 kb; they belong to the order nidovirales, family coronaviridae, and subfamily coronavirinae (woo et al. 2012) . based on antigenic relationships, the classification of coronaviruses was traditionally divided into 3 genera, but they were replaced by the following four genera: alphacoronavirus ( a l p h a -c o v ) , b e t a c o r o n a v i r u s ( b e t a -c o v ) , gammacoronavirus (gamma-cov), and deltacoronavirus (delta-cov), which are based on genetic and antigenic characteristics (woo et al. 2010 ). epidemiological surveys have indicated that bats and birds seem to be natural reservoirs for alpha-and beta-covs and gamma-and delta-covs, respectively (bolles et al. 2011; woo et al. 2012) . covs in four genera have been verified in a variety of species, e.g., canines, felines, and birds (chan et al. 2013) . six covs have been identified in swine (table 1) : porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), swine acute diarrhea syndrome coronavirus (sads-cov), and porcine respiratory cov (prcov) in the alpha-cov genus, porcine hemagglutinating encephalomyelitis virus (phev) in the beta-cov genus, and porcine deltacoronavirus (pdcov) in the delta-cov genus (jung et al. 2016; pan et al. 2017; pensaert and de bouck 1978; woo et al. 2012; zhang 2016; zhou et al. 2018 ). among the six swine covs, tgev, pedv, pdcov, and sads-cov are enteric viruses that cause diarrhea in the pig population, resulting in significant economic losses and tremendous threats to the pig industry worldwide. the four swine enteric covs causing highly contagious enteric diarrhea in neonatal and suckling piglets are clinically characterized by vomiting, watery diarrhea, dehydration, and high morbidity and mortality (gong et al. 2017; hsu et al. 2018) . because the clinical signs of pigs infected by these covs are very similar (table 1) , it is difficult to differentiate the specific pathogens based on clinical symptoms. effective high-throughput detection methods are needed for their differential determination and would represent powerful tools to prevent and control diseases. as far as we know, many standard detection methods can be used to distinguish between causative agents, including virus isolation, electron microscopy, virus neutralization, and indirect immunofluorescence assays. however, these methods are time-consuming, laborious, and not suitable for the early and rapid detection of the four swine enteric covs (carman et al. 2002; dulac et al. 1977; van nieuwstadt et al. 1988 ). the enzyme-linked immunosorbent assay is a powerful and highthroughput method for detecting specific antibodies, but the immune system of piglets is not well developed, so serological methods for detecting antibodies against these viruses are also not suitable for rapid and early detection. polymerase chain reaction (pcr) methods have been widely used to detect pathogens since the pcr was invented; pcr has proven to be powerful and convenient tools for precise detection of diarrheal pathogens in pig populations (ben salem et al. 2010; collins et al. 2008; kim et al. 2007 ). this paper reviews various pcr-based methods for the rapid and efficient detection of these pathogenic covs in swine intestines. pan-cov rt-pcr assay for the detection of covs figure 1 summarizes the basic workflow for the detection of the swine enteric coronaviruses from clinical samples. generally, porcine fecal or intestinal samples need to be suspended and homogenized in sterile pbs and centrifuged to remove debris. the supernatant should be collected and filtered through a 0.45-μm filter to remove the debris and some potential bacteria. the yield supernatant can be used to extract the total rnas by trizol reagent or rna extraction kit. the total rnas are used to reverse transcription by random primers to generate cdna. this cdna is employed as a template to do pcr amplification either by specific individual coronavirus or by pan-cov primers first then followed by specific primers targeting individual swine enteric coronavirus when necessary. the pcr products are eventually subjected to dna electrophoresis and analyzed under uv light to identify the desired bands. the pan-cov pcr method is a powerful tool for detecting all known and unknown covs; it is based on the conserved gene sequences among them (moes et al. 2005 ). this assay is widely used to detect covs. pan-cov rt-pcr was employed to detect all known covs in the human respiratory tract (vijgen et al. 2008) and to detect distinct alpha-covs in five different bat species (escutenaire et al. 2007; lazov et al. 2018; vijgen et al. 2008) . for swine enteric covs, pan-cov pcr also played an important role in identification. during the early stage of investigating cases of diarrhea in piglets in the usa caused by the pedv variant and pdcov, pan-cov rt-pcr was applied to identify the causative agent together with electron microscopy and sequencing stevenson et al. 2013 ). in addition, during the identification of piglet diarrhea disease caused by sads-cov in china, pan-cov rt-pcr was employed (pan et al. 2017) . in 2018, hu et al. reported an improved one-step pan-cov rt-pcr (hu et al. 2018) . though pan-cov pcr could detect all known covs in humans and animals, it could not make a differential detection, so it is not suitable for routine swine enteric cov detection. porcine epidemic diarrhea (ped) is an enteric disease caused by pedv that can infect pigs of all ages with different levels of clinical signs of vomiting, diarrhea, dehydration, and weight loss, but the disease is much more severe in suckling piglets (have et al. 1992; shibata et al. 2000; song and park 2012; sueyoshi et al. 1995) . ped was first reported in england in 1971, but the pedv was isolated for the first time in belgium in 1978 (pensaert and de bouck 1978; song and park 2012) ; however, the epidemic was not controlled in europe before 2000. in china, pedv was first identified in the 1980s, after which it was reported in some asian countries, e.g., japan and korea (kusanagi et al. 1992; song and park 2012; takahashi et al. 1983 ). in october 2010, a severe ped outbreak caused by a highly virulent pedv variant emerged in southern china with high mortality ranging from 70 to 100%; the result was devastating damage to the pig farm industry and tremendous economic losses, and later, the pedv variant spreads to other countries, e.g., usa, canada, and mexico ( for the early and rapid detection of pedv, different types of pcr methods have been developed. a real-time reverse transcription recombinase polymerase amplification assay (rt-rpa) based on the nucleocapsid gene of pedv was reported in 2018; the assay was able to detect 23 copies per reaction and was performed for 20 min at 40°c . based on the advantage of increased thermal conductivity of solid gold nanometal particles that could reduce nonspecific amplification and increase specific amplification, yuan et al. developed a nanoparticle-assisted pcr assay for the detection of pedv on the basis of the n gene in 2015, and the assay could detect 2.7 × 10 −6 ng/μl of pedv rna (yuan et al. 2015) . ren and li designed six primers according to the sequence of the n gene and established a reverse transcription loop-mediated isothermal amplification (rt-lamp) for the rapid detection of pedv (within 50 min) at 63°c. the detection limit of the method was 1 × 10 −4 μg pedv rna per reaction (ren and li 2011) . wang et al. designed five primers on the basis of the n gene sequence of pedv and established a reverse transcription cross-priming amplification-nucleic acid test strip (cpa-nast) for the detection of pedv. the method had high specificity for the detection of pedv and had the same sensitivity as traditional rt-pcr; the detection limit was a 10 −6 dilution of plasmid containing the target gene ). xing et al. developed an rna extraction and transcription-free, nanoparticle-based pcr (nbp-pcr) method to specifically detect pedv, and the sensitivity of the schematic diagram of the workflow for swine enteric coronavirus detection by rt-pcr. porcine fecal or tissue samples are homogenized in sterile pbs and centrifuged to remove debris. the supernatant is filtered through a 0.45-μm filter and used to total rna extraction. the total rnas are subjected to reverse transcription using random primer to generate cdna. with this cdna as template, the pcr amplification is carried out either by specific individual coronavirus or by pan-cov primers first then followed by specific primers targeting individual swine enteric coronavirus when necessary. the pcr products are subjected to dna electrophoresis and observed under uv light to identify the desired bands the method was 400-fold higher than that of conventional rt-pcr. in 153 fecal samples, the positive detection rate of nbp-pcr specific was much higher than that of conventional rt-pcr (5.88%) and sybr green real-time rt-pcr (xing et al. 2016 ). zhou et al. developed a conventional rt-pcr method, an sybr green i real-time rt-pcr, and taqman real-time rt-pcr reagents to detect the highly conserved m gene of pedv; the detection limit of the taqman real-time rt-pcr was 10 copies/μl of the target gene, and the sensitivity of the taqman real-time rt-pcr was 100-fold and 10,000-fold higher than that of sybr green i real-time rt-pcr and conventional rt-pcr, respectively ). in addition, there were variant pedv strains circulating in the field and the pcr methods for differentiating them had been established. song et al. analyzed pathogenicity and immunogenicity of pedv strain designated dr13 in piglets, which was a highly vero cell-adapted virus and could be employed as a vaccine candidate, and applied a restriction fragment length polymorphism (rflp) assay to differentiate dr13 from wild-type virus based on the difference of open reading frame (orf) 3 sequence (song et al. 2003) . in 2008, lee et al. reported an rt-pcr-based rflp assay targeting the n gene of pedv to distinguish field strains of pedv in korea from an attenuated-live vaccine j-vac, which was used in pig population to prevent pedv . for differentiation between attenuated-type pedvs including attenuated dr13, kped-9, and p-5v that were used as live virus vaccine and wild-type pedvs including cv777, brl/ 87, lzc, parent dr13, and field samples, park et al. established an rt-pcr assay based on the difference of orf 3 gene sequence of attenuated-and wild-type pedv, in which 51 nucleotide deletions were found in all live pedv vaccine . in may 2013, the virulent strain of pedv was verified in the usa resulting in significant economic losses in the swine industry. and a variant strain (oh851) of pedv emerged in the usa in december 2013, which differs from the virulent strains of pedv in the nucleotides of the 5′end of spike gene. to differentiate these two genotypes of pedv circulating in the usa, wang et al. reported a duplex probe-based real-time rt-pcr targeting the difference of spike gene among virulent and variant strains (wang et al. 2014b ). sequence analysis of pedv genome indicated that pedv attenuated vaccine strains (e.g., the cv777 and zj08 in china, the p-5v in japan, and kped-9 and dr13 in south korea in asia) have 49 base pair deletion in the open reading frame 3 (orf3); for differentiation of these cell-adapted vaccine strains from field strains, zhu et al. developed a nanoparticle-assisted rt-pcr assay targeting the orf3 (zhu et al. 2016b) . because three major pedv types have been identified in the usa after 2013 including the original us pedv strains, the spike gene insertion-deletion pedv strains, and the pc177 strain that possess a 197 amino acid-deletion in the s1region of spike protein, liu and wang developed a reverse transcription-pcr method to differentiate these variants on the basis of differences in the s1 gene (liu and wang 2016) . since 2010, pedv variants with base deletions and insertions in the s gene emerged in china and caused significant losses in piglets; zhao et al. developed a taqman probe-based real-time pcr method for the detection of different pedv variants and classical pedv strains based on the sequence difference of the pedv s gene, and the detection limit of the method was 5 × 10 2 target gene copies (zhao et al. 2014 ). su et al. established a duplex taqman probe real-time rt-qpcr method for detecting and differentiating classical and variant pedvs targeting the difference in the s gene; the detection limit of the method was 4.8 × 10 2 genome copies/reaction for both the classical and variant pedv. the results of clinical sample detection showed that the assay was more sensitive than conventional pcr, and variant pedv was prevalent in china (su et al. 2018) . due to the wide use of a live attenuated pedv vaccine, classical and wild variant strains circulating in pig farms are common; therefore, he et al. established a multiplex rt-pcr to differentiate these strains based on the difference in s gene sequences, and the detection limit of the method was 1 × 10 1.7 tcid 50 /100 μl for pedv (he et al. 2019 ). due to variant pedv strains that emerged in china since 2010 and attenuated pedv vaccines (e.g., cv777 strain) being widely used in china, liu et al. reported a taqman probe-based realtime pcr to differentiate these virulent strains and attenuated vaccine strains based on the orf3 deletion region to detect virulent pedv strains in vaccinated pig population (liu et al. 2019b ). transmissible gastroenteritis (tge) caused by tgev is an acute enteric diarrhea disease in pigs (garwes 1988) . tgev was isolated for the first time in 1946, and then outbreaks of the virus occurred in many countries in america, asia, and europe (doyle and hutchings 1946; kim et al. 2000; stevenson et al. 2013) . tgev causes severe enteritis in piglets before weaning, and the clinical signs include diarrhea, vomiting, dehydration, and high mortality, resulting in significant economic losses (ding et al. 2017; penzes et al. 2001; saif 1999) . for the early and rapid detection of tgev, different types of pcr-based methods have been established. chen et al. designed six specific primers targeting the nucleocapsid gene of tgev and developed an rt-lamp assay for the detection of tgev, which involved incubation at 60°c for 1 h. the sensitivity of rt-lamp was comparable to that of nested pcr described by rodríguez et al. in 2008 , and it was 10 times more sensitive than the pcr reported by paton et al. in 1997, which could detect 10 pg of rna per reaction paton et al. 1997; rodríguez et al. 2008) . rt-lamp for the detection of the tgev targeting the n gene sequence by incubation at 60°c for 30 min was reported by li et al., and the sensitivity of rt-lamp was much higher than that of a gel-based rt-pcr kit purchased from haigene company, china (li et al. 2012 ). vemulapalli et al. described a taqman probe-based real-time rt-pcr that specifically amplified conserved s gene sequences. the detection limit of the method was 1 tcid 50 /ml of tgev rna. the sensitivity of the assay was higher than that of the nested rt-pcr assay reported by kim et al. (kim et al. 2000; vemulapalli et al. 2009 ). wang et al. developed a rapid detection of tgev using real-time reverse transcription recombinase polymerase amplification (rt-rpa) based on the spike gene, and the method could detect 100 copies of tgev rna after 20 min at 40°c conditions . a real-time rt-pcr assay described by vemulapalli et al. was compared with the rt-rpa method in the analysis of 76 clinical samples, and the two methods generated the same results (vemulapalli et al. 2009; wang et al. 2018 ). in the mid-1980s, prcov, which has a deletion of nucleotides of the s gene that is present in tgev (laude et al. 1993) , was reported in europe, north america, and asia (pensaert et al. 1986; wesley et al. 1990 ). paton et al. developed an rt-pcr assay to differentiate tgev and prcv based on the difference in the s gene of the two viruses (paton et al. 1997) . pdcov is a novel swine enteric diarrhea virus that causes severe diarrhea, vomiting, and dehydration in piglets (janetanakit et al. 2016; song et al. 2015) . pdcov was first discovered in samples from the healthy pig that were collected in 2009 in hong kong when a molecular surveillance study was performed (woo et al. 2012) . in february 2014, the virus was detected in piglets with severe diarrhea in the usa (marthaler et al. 2014; wang et al. 2014a ). subsequently, pdcov was reported in south korea, mainland china, and thailand (chen et al. 2015a; janetanakit et al. 2016; lee and lee 2014; song et al. 2015) . to detect the virus with precision, pcr-based methods have been created. in 2014, pigs on 5 farms in ohio, usa, had clinical diarrheal disease. wang et al. developed a onestep rt-pcr targeting membrane and nucleocapsid gene of delta-cov and determined the causative agent as porcine delta-covs, but they did not evaluate the detection limit of the assay (wang et al. 2014a ). in 2015, song et al. established a nested rt-pcr method on the basis of the nucleocapsid gene sequence of the pdcov hku15 strain to identify the causative agent causing acute diarrhea in a pig farm in jiangxi, china, and the sensitivity of the assay was also not determined . to analyze the characteristics of porcine delta-covs in the usa, ma et al. established a real-time rt-pcr method for specific detection of the n gene, but the sensitivity of the assay was also not determined (ma et al. 2015) . for the analysis of pathogenicity and pathogenesis of a porcine delta-cov cell culture isolate, chen et al. developed an m gene-based real-time pcr method for detecting viral titers in different organs, but the sensitivity of the assay was not described in the publication (chen et al. 2015b ). marthaler et al. developed a one-step probe-based real-time rt-pcr method targeting the m gene sequence of pdcov, which was applied by the university of minnesota veterinary diagnostic laboratory. the detection limit of the assay was 2 viral rna copies per reaction, but the specificity of the assay was not evaluated (marthaler et al. 2014 ). the commercial reverse transcription-insulated isothermal pcr (rt-iipcr) pockit™ methods (pockit™ pedv reagent set and pockit™ pdcov reagent set, genereach usa, lexington, ma, usa) was used to analyze pedv and pdcov coinfection that occurred in diarrhea disease; zhang et al. described two singleplex rt-iipcr tests and a duplex real-time rt-pcr test for the detection of pedv and pdcov that were based on targeting the conserved m gene sequence. the detection limits of singleplex rt-iipcr were 21 rna copies per reaction for pedv and 9 rna copies per reaction for pdcov, and those of the duplex rt-iipcr were 7 rna copies and 14 rna copies per reaction for pedv and pdcov, respectively ). sads-cov, also designated seacov or peav, is a novel porcine enteric diarrhea virus that can cause severe and acute diarrhea and rapid weight loss in piglets (pan et al. 2017; zhou et al. 2018; zhou et al. 2017) . sads-cov was identified for the first time in southern china in late 2017, and it caused more than 24,000 piglet deaths, resulting in significant economic losses (gong et al. 2017) . the clinical signs of infection with sads-cov are similar to those of other known swine enteric covs: tgev, pedv, and pdcov (dong et al. 2015; sun et al. 2016) . for specific detection of sads-cov, zhou et al. established an sybr premix ex taqii-based real-time pcr on the basis of the rna-dependent rna polymerase (rdrp) gene for the detection of sads-cov; the sensitivity of which was not evaluated ). zhou et al. developed a taqman-based real-time pcr assay for sads-cov detection based on the conserved sequence within the n gene; the detection limit of the assay was 3.0 × 10 1 copies/μl, and the sensitivity of the method was 10-fold higher than that of conventional pcr, which also targeted the n gene (zhou et al. 2018 ). because the clinical signs caused by the four enteric covs in piglets are similar to each other, it is very difficult or timeconsuming to make a clear diagnosis of mixed infection in pigs using a single pcr method. therefore, it is critical to developing a multiplex polymerase chain reaction (mpcr) in which two or more loci can be simultaneously detected in the same reaction (chamberlain et al. 1988 ). tgev and pedv are traditional swine enteric diarrheal viruses, so several mpcr methods that can differentially detect the two viruses have been previously established. in 2001, kim et al. reported a duplex rt-pcr assay to detect tgev and pedv in one pcr tube targeting the s gene of the two viruses, and the detection limit of the assay was 2 tcid 50 /200 μl (kim et al. 2001) . in 2007, kim et al. established a multiplex realtime rt-pcr method based on the nucleocapsid (n) gene for the simultaneous detection and quantification of tgev and pedv, and the detection limits of this method were 90 copies and 70 copies for tegv and pedv, respectively (kim et al. 2007 ). zhu et al. designed two pairs of primers on the basis of the n gene sequences of tgev and pedv and established a nanoparticle-associated pcr assay; the sensitivity of the assay was 10-fold higher than that of conventional pcr (zhu et al. 2017) . apart from the four enteric covs, several enteric diarrheal viruses have been discovered, including porcine sapovirus (psav), porcine norovirus (pnov), porcine teschen virus (ptv), porcine kobuvirus (pkv), seneca valley virus (svv), porcine rotavirus (prv), porcine reovirus (reov), porcine bocavirus (pbov), and porcine astrovirus (pastv). therefore, some mpcr methods have been created to detect swine enteric diarrheal viruses, but they are not limited to enteric covs (ding et al. 2019) . ben salem et al. developed a nested rt-pcr method for the detection of pedv, tgev, and prv based on the sequence of the tgev purdue strain (accession no. nc_002306), the pedv strain cv777 (accession no. nc_003436), and the nsp5 gene of the prv-a osu strain (accession no. x15519), respectively. the detection limits of multiplex nested rt-pcr for tgev and pedv were 10 2 tcid 50 /ml and 27.2 μg/μl of rna, respectively (ben salem et al. 2010) . in 2013, zhao et al. developed a multiplex rt-pcr assay to identify pedv, tgev, prv-a, and porcine circovirus 2 (pcv2) in one reaction, and the sensitivity of the assay for the detection of tgev and pedv was 10-fold lower than that of a single rt-pcr method ). zhao et al. established a multiplex rt-pcr assay for rapid and differential diagnosis of pedv, tgev, prv-a, and pcv2 targeting the s gene, segment 6 region, and orf2 sequence, and the detection limits of the assay for tgev and pedv were 1.74 × 10 4 and 2.1 × 10 3 copies, respectively ). liu et al. developed a multiplex pcr assay to detect five diarrhea-related pig viruses: pedv (nucleoprotein), tgev (spike glycoprotein), prv-a, porcine group c rotaviruses (prv-c), and pcv2. the detection limits of the assay for pedv and tgev were 5 copies per reaction (liu et al. 2019a ). wen et al. developed a multiplex real-time pcr method based on evagreen fluorescent dye to simultaneously detect and distinguish pedv-nucleoprotein (n), tgev-spike glycoprotein (s), prv-a, prv-c, and pcv2, and the limits of detection ranged from 5 to 50 copies/μl (wen et al. 2019 ). there was no multiplex pcr method exclusively for differential detection of the four enteric covs until huang et al. developed a taqman-probe-based real-time rt-pcr method in 2019 targeting the m gene of pedv, the n gene of tgev, the m gene of pdcov, and the n gene of sads-cov. their multiplex real-time rt-qpcr assay could detect 10-100 copies of each target gene per pathogen (huang et al. 2019 ). as mentioned above, nested rt-pcr, rt-rpa, nanoparticleassisted pcr, rt-lamp, cpa-nast, sybr green-based real-time pcr, evagreen-based real-time pcr, and taqman probe-based real-time pcr represent single or multiplex pcr methods that have been developed to detect one, two, or four enteric pathogenic covs. in the field, the causative agents of swine enteric diarrhea are mixed; single rt-pcr methods are not suitable for rapid and efficient detection of covs even though they have higher sensitivity than multiplex rt-pcr. in addition, the pcr fragments of some single rt-pcr methods have to be subjected to agarose gel analysis to determine results, which is time-consuming. in particular, nested rt-pcr requires two pcr steps, resulting in an increased likelihood of contamination, so this method has not been widely used for pathogen detection. for rapid and efficient detection of pathogenic swine enteric covs, multiplex pcr methods, including conventional multiplex rt-pcr and multiplex real-time rt-pcr, are ideal options. although conventional multiplex rt-pcr can simultaneously differentially detect several different pathogens in one reaction, the method also possesses the disadvantages of single rt-pcr, e.g., risk of product contamination, and the inability to monitor developments in real time. the sensitivity of rt-pcr is 10-100 times lower than that of real-time rt-pcr, and the viral loads cannot be measured (keyaerts et al. 2006) . recently, real-time taqman probe-based rt-pcr methods have become increasingly used to detect targets because they own many advantageous characteristics: the ability to perform differential detection, high specificity, high sensitivity, high-throughput ability, high repeatability, quantification ability, and the ability to assess results in real time (slavov et al. 2016; teng et al. 2015; zhu et al. 2016a ). therefore, huang et al. in our lab developed a taqman-probe-based real-time rt-pcr for the differential detection of pedv, tgev, pdcov, and peav (huang et al. 2019) . although real-time taqman probe-based rt-pcr possesses many merits and can be used to rapidly and efficiently detect pathogenic swine enteric covs, the method requires a high-precision and sophisticated instrument, practical technicians, and a good laboratory; therefore, it cannot be used for detection in under-equipped laboratories or on-site. rapid, accurate, and more practical detection methods are of great significance for the surveillance, prevention, and control of enteric diseases in pigs, so novel assays are still deserved further development. for instance, test strip detection methods, which can be used by under-equipped laboratories or on-site and can be easily operated to quickly generate results, are urgently needed. as mentioned earlier, apart from the four enteric covs, many other pathogens causing diarrhea in pigs have been identified. and the causative agents of swine enteric diarrhea are mixed in the field. to rapidly determine whether the pathogens are enteric covs, pan-cov pcr is the best option to initially detect from clinical samples and followed by specific primers targeting individual swine enteric coronavirus for further identification when the result from pan-cov pcr is positive. however, when the pan-cov pcr results are negative, specific primers targeting other enteric diarrheal viruses are required to determine the causative agents. authors' contributions yf and gl conceived the review. fy and bl collected all references and wrote the manuscript draft. gl edited the manuscript. conflict of interest the authors declare that they have no competing interests. ethical statement no ethical approval was required as this is a review article with no original research data. multiplex nested rt-pcr for the detection of porcine enteric viruses sars-cov and emergent coronaviruses: viral determinants of interspecies transmission field validation of a commercial blocking elisa to differentiate antibody to transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus and to identify tgev-infected swine herds deletion screening of the duchenne muscular dystrophy locus via multiplex dna amplification interspecies transmission and emergence of novel viruses: lessons from bats and birds complete genome sequence of a variant porcine epidemic diarrhea virus strain isolated 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conventional rt-pcr, sybr green i and taqman real-time rt-pcr assays for the detection of porcine epidemic diarrhea virus development of taqman real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus a sensitive duplex nanoparticle-assisted pcr assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens establishment of a nanoparticle-assisted rt-pcr assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-026729-hn0q0sbv authors: xu, jun; xia, kai; li, pinyi; qian, chenggong; li, yudong; liang, xinle title: functional investigation of the chromosomal ccdab and hipab operon in escherichia coli nissle 1917 date: 2020-06-13 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10733-6 sha: doc_id: 26729 cord_uid: hn0q0sbv toxin-antitoxin systems (tass) have attracted much attention due to their important physiological functions. these small genetic factors have been widely studied mostly in commensal escherichia coli strains, whereas the role of tass in the probiotic e. coli nissle 1917 (ecn) is still elusive. here, the physiological role of chromosomally encoded type ii tass in ecn was examined. we showed that gene pair ecolin_00240-ecolin_00245 and ecolin_08365-ecolin_08370 were two functional tass encoding ccdab and hipab, respectively. the homologs of ccdab and hipab were more conserved in e. coli species belonging to pathogenic groups, suggesting their important roles in ecn. crispri-mediated repression of ccdab and hipab significantly reduced the biofilm formation of ecn in the stationary phase. moreover, ccdab and hipab were shown to be responsible for the persister formation in ecn. biofilm and persister formation of ecn controlled by the ccdab and hipab were associated with the expression of genes involved in dna synthesis, sos response, and stringent response. besides, crispri was proposed to be an efficient tool in annotating multiple tass simultaneously. collectively, our results advance knowledge and understanding of the role of tass in ecn, which will enhance the utility of ecn in probiotic therapy. key points • two tass in ecn were identified as hipab and ccdab. • knockdown of hipab and ccdab resulted in decreased biofilm formation of ecn. • transcriptional silencing of hipab and ccdab affected the persister formation of ecn. • an attractive link between tass and stress response was unraveled in ecn. • crispri afforded a fast and in situ annotation of multiple tass simultaneously. electronic supplementary material: the online version of this article (10.1007/s00253-020-10733-6) contains supplementary material, which is available to authorized users. nowadays, a variety of probiotics have been explored and applied to food and therapeutic interests, of which lactic acid bacteria biologics are the dominant types due to their biological functions and safety. among the enteric commensal escherichia species, e. coli nissle 1917 (ecn) is the only one which has been authorized as a probiotic and applied to treat various human gastrointestinal disorders and infections, including diarrhea, diverticulitis, and inflammatory bowel disease (ibd) (secher et al. 2017; sonnenborn 2016) . furthermore, as a safe delivery vector, ecn has been successfully developed to upload various functional allergens in the allergy desensitization, wherein its probiotic functions have been extended synergistically due to the immunotherapy line (sarate et al. 2018) . in a colorectal cancer chemoprevention test, the recombinant ecn was directed specifically to the cancer cell surface and in situ expressed myrosinase to perform the host-ingested glucosinolate hydrolysis for sulphoraphane production (ho et al. 2018) . besides, genetically engineered ecn strains have been used in vaccine and pharmaceutical preparations (chaudhari et al. 2017; ou et al. jun xu and kai xia contributed equally to this work. electronic supplementary material the online version of this article (https://doi.org/10.1007/s00253-020-10733-6) contains supplementary material, which is available to authorized users. 2016). when the ecn is employed for probiotic therapy, the viability of ecn is one important factor that determines the final therapeutic effect, therefore, to sustain the survival of ecn on a desired level is necessary during probiotic therapy. it is rational that forming biofilm and persister cells will promote population colonizing onto the gastrointestinal tract mucosal layer, thereby allowing probiotics to in situ develop and carry out functional therapies. previous studies showed that biofilm formation promotes the ability of lactobacillus strains in resistance to temperature, gastric ph and mechanical forces (salas-jara et al. 2016) , and a bile-induced biofilm formation during stationary growth allows bifidobacteria strains for strong colonization in the gastrointestinal tract (ambalam et al. 2014) . it is evident that the probiotic mechanism of ecn is inseparable from its colonization ability (hancock et al. 2010) . beyond the contribution of pili (f1a, f1c, and curly fimbriae), iron chelating carriers, and uptake systems, the better capacity of ecn in biofilm formation competitively inhibits adhesion and invasion of pathogenic e. coli to intestinal epithelial cells (dembinski et al. 2016; jiang et al. 2014) . persister cells firstly reported in the early 1940s are phenotypic variants in a given population that presents antibiotic tolerance due to their slow or non-growing state (page and peti 2016) . persister cells are able to revert to normal growth when the adverse stimuli such as antibiotics are removed. as studies progressed, forming persister cells has been generally accepted as an important strategy for bacteria to survive upon hostile environments (page and peti 2016; van den bergh et al. 2017) . moreover, the persistence phenomenon has been widely described and determined in e. coli. therefore, it is likely that cells in a persistent state will be helpful for ecn to become the winner during outcompeting with the pathogenic enteric microbiome in the intestine. however, the current understanding of the underlying mechanisms involved in the biofilm and persister formation of ecn is inadequate. multiple signaling layers have been reported to participate in modulating biofilm and persister formation, including stringent control (fujita et al. 2017 ), quorum sensing system (li et al. 2018 ), c-di-gmp signaling pathway (moreira et al. 2017) , and toxin-antitoxin system (tas) (sun et al. 2017 ). among these, tas has attracted much attention due to their critical roles in cell physiologies such as programmable cell death, biofilm formation, and persister formation (harms et al. 2018; page and peti 2016) . a typical tas constituted a bicistron line which is composed of a stable toxin or growth arrest factor and its unstable inhibitor (antitoxin) (yang and walsh 2017) . currently, six types of tass have been classified, wherein the distribution and prevalence of type ii are the most dominant and universal in bacteria and archaea (page and peti 2016) . in e. coli, more than ten kinds of type ii tass have been studied, of which the f-plasmid-based ccdab and high persistence allele hipab are two of the best characterized and identified that are mainly responsible for the plasmid maintenance and persister formation, respectively (ogura and hiraga 1983; semanjski et al. 2018; wen et al. 2014 ). however, the relevant knowledge in ecn is still insufficient. in addition to their critical roles in physiologies, tass have recently been developed as an efficient counterselectable marker in e. coli recombineering. for example, the toxic gene ccdb is developed as a counterselection module to improve seamless mutagenesis and the efficiency of high-throughput, restriction-free cloning, and screening process in e. coli (wang et al. 2014; lund et al. 2014) . endogenous tas ydce-ydcd has been used for constructing a stable and foodgrade expression system in bacillus subtilis . these shed light on the application of endogenous tass in the genetic modification of ecn in terms of plasmid construction and chromosomal gene knock-in. in the current study, we identified two type ii tas pairs and investigated their physiological roles in ecn. to achieve this, the well-known gene-editing tool crispr interference (crispri) is first introduced to annotate tas. we demonstrated that the identified ccdab and hipab are associated with the formation of biofilm and persister cells in ecn. our findings highlight that tass may be an important candidate in the in situ engineering of ecn when employed as probiotic therapy to sustain cell viability, and crispri will be an efficient and robust tool in driving the engineering process. the strains and plasmids used in this study were listed in table 1 . all strains were grown in lb medium (10 g/l peptone, 5 g/l yeast extract, 10 g/l nacl) at 37°c, with shaking at 180 r/min (liquid culture). for plasmid maintenance, lb medium was supplemented with ampicillin (50 μg/ml), kanamycin (50 μg/ml), and chloramphenicol (25 μg/ml) when appropriate. e. coli dh5α and bl21 (de3) were applied for plasmid propagation and preparation, and protein expression, respectively. putative type ii tass in the e. coli genome were predicted by a web-based tool ta finder (http://202.120.12.133/tafinder/ index.php) using default parameters. comparative analysis of the tass in ecn with other e. coli species was performed using the blast tool involved in ncbi (https://www.ncbi. nlm.nih.gov/). the synteny analysis of tass in each e. coli strains was carried out by synttax (http://archaea.u-psud.fr/ synttax/). bprom was used for the prediction of putative promoters (http://www.softberry.com/). the homologous sequences of ecolin_00240, ecolin_00245, ecolin_ 08365, and ecolin_08370 from other e. coli groups were used for the construction of phylogenetic trees via mega6.0 software (tamura et al. 2013) . the evolutionary history was determined using the neighbor-joining method and the pcloning and expression of putative toxin and antitoxin genes putative toxin and antitoxin genes were amplified by pcr using the ecn genome as the template and prime star max dna polymerase (takara, japan), with the primers listed in table s1 (p1-p8, supplementary materials). the obtained dna fragments were inserted into the multiple cloning sites mcs1 and mcs2 included in petduet-1 (novagen) after double digestion by appropriate restriction enzymes (bamhi and psti for gene encoding toxin, and kpni and ecorv for gene encoding antitoxin), respectively, to produce plasmid comprising gene encoding either toxin or antitoxin protein or both. positive colonies were selected by colony-pcr using primer p9-p12 (table s1 ) and verified by sequencing. evaluation of the toxicity of toxin proteins was performed as previously described with some modifications (xia et al. 2019) . shortly, overnight cultures of e. coli bl21 (de3) comprising different plasmids were inoculated to fresh lb medium with 1% inoculum and grown until an od 600 of 0.25 ± 0.05. then, 1 mm iptg was added and cell growth was monitored by measuring od 600 of the cultures. after induction for 4 h, the aliquots of each culture were 10-fold serially diluted and spotted on plates without iptg to detect cell viability. all primers were synthesized by sangon biotech (shanghai) co., ltd. in crispri system, the nuclease-deficient cas9 (dcas9) was expressed using an anhydrotetracycline (atc) inducible promoter, while the single-guide rna (sgrna) was expressed continuously with promoter j23119 . in order to inhibit the expression of ccdab and hipab in vivo, two specifically designed sgrnas comprising 20-nucleotide (nt) spacer sequence (n20 sequence) complementary to the target genes ccdb and hipb were designed by a web-based tool (http://crispr.dbcls.jp/), respectively. to improve the efficiency, six n20 sequences possessing the best hits were selected and synthesized in the form of primers (three for each gene) (table s1 , sequence underlined, only one best n20 sequence was shown). the detailed processes of construction were shown in supplementary materials (fig. s1a) . briefly, dna fragments containing the n20 sequence were amplified by pcr using plasmid pgrna-bacteria as the template. the obtained dna fragments were purified and sub-cloned into plasmid pgrna-bacteria between spei and bamhi restriction sites to produce plasmid pgrcb and pgrhb. the positive colonies were verified by sequencing. the plasmid pgrcb and pgrhb were transformed into ecn along with pdcas9bacteria to create the respective ccdab and hipab knockeddown strains (fig. s1b) . to repress the expression of ccdab and hipab, 2 μm atc (sigma, usa) was added to the culture medium at the indicated time. the repression of gene expression was confirmed by rt-pcr. to suppress the transcription of ccdab and hipab simultaneously, the plasmid purcbh comprising the sgrna targeting to ccdb and hipb was constructed and transformed along with pdcas9-bacteria into ecn (fig. s2 ). o v e r n i g h t c u l t u r e s o f e c n : : p d c a s 9 : : p g r c b , ecn::pdcas9::pgrhb , and ecn::pdcas9::purcbh were inoculated to fresh lb medium with 1% inoculum (2 μm atc was added to the medium where indicated) and grown until od 600 of 0.6 ± 0.05, respectively. cell samples were collected by centrifugation (8000 r/min, 5 min). rna extraction was carried out using takara minibest universal rna extraction kit (takara, japan) according to the manufacturer's instructions. the rna yield and quality were evaluated with a nanodrop uv spectrometer (thermo scientific, usa). the first-strand cdna was synthesized using the primescript™ii 1st strand cdna synthesis kit (takara, japan). the purified cdna fragments were used as the template in the following polymerase chain reaction (pcr) analysis. for quantitative reverse transcription (qrt-pcr) analysis, the rna samples were prepared similarly. shortly, overn i g h t c u l t u r e s o f e c n , e c n : : p d c a s 9 : : p g r c b , ecn::pdcas9::pgrhb, and ecn::pdcas9::purcbh were inoculated to fresh lb medium containing 2 μm atc with 1% inoculum and grown for 24 h. then, cells were collected by centrifugation and used for rna extraction and cdna synthesis as described above. the qrt-pcr was performed with the sybr premix ex taq™ii kit (takara) with a final volume of 20 μl, which contained 10 μl of sybr premix ex taq ii, 0.4 μl of each primer (final concentration 0.4 μm), 2 μl of cdna, and 7.2 μl of rnase-free dh 2 o. all of the primers used in this part were listed in table s1 (p23-p42). the amplification procedure included denaturation at 95°c for 5 min; 40 cycles of 95°c for 15 s, and 60°c for 30 s. the melting curves were analyzed by taipu tib8600 qrt-pcr system (taipu, china). the data were analyzed by the δδct method using 16s rrna as an internal reference. the relative fold change was calculated from 2 −δδct , in which, δct = ct target gene -ct internal reference gene, δδct = ct sample -ct control. the assay of biofilm formation was performed as previously reported with some modifications (sun et al. 2017) . in brief, 50 μl aliquots of each culture were inoculated into 5 ml fresh lb medium (with or without atc) contained in two different 6-well polystyrene culture plates (final od 600 of~0.05), followed by incubating at 37°c without shaking for 24 h. then, the medium was discarded, and the wells were gently rinsed with 1x pbs to remove planktonic and loosely adhered cells. the adhered biofilm was quantified by staining the cells with 1% crystal violet solution for 30 min at room temperature. the excess crystal violet was removed by washing the wells three times with distilled water. crystal violet bound to the adhered cells was solubilized in 1 ml of 95% ethanol and quantified by measuring absorbance at 540 nm and 620 nm. for biofilm assay using a differential interference microscope (olympus u-lh100-3, japan), the sterilized coverslips were added to the well after inoculation as described above. after incubation for 24 h, the coverslips were taken out and washed gently using pbs buffer (0.15 m, ph 7.2) to remove the planktonic cells. the biofilm formation of each tested group was captured using the microscope. all experiments were performed with three independent biological replicates. all antibiotics (usp grade) were purchased from sangon biotech (shanghai) co., ltd. the minimal inhibitory concentration (mic) of gentamycin and norfloxacin was detected as previously reported (andrews 2001) . briefly, overnight cultures of different strains were diluted by 1:100 into fresh lb medium containing gentamycin or norfloxacin with different concentrations. the mixtures were added to 96-well plates (200 μl in each well) and incubated at 37°c for 24 h. the optical density (od 600 ) was determined by a microtiter plate reader (victor™x3, perkinelmer). the lowest antibiotic concentration that inhibited visible bacterial growth was defined as the mic (od change < 0.05). for persistence level detection, a time-dependent killing curve was assayed. in brief, overnight cultures of different strains were inoculated into fresh lb medium with or without atc (2 μm) and grown for 24 h, followed by adding gentamycin or norfloxacin to a final concentration of 10-fold the mic. following treatment, cells collected at indicated time by centrifugation (8000 r/min, 3 min) were washed and resuspended using a fresh lb medium. the suspension was then serially diluted and plated on lb agar plates to obtain the colony-forming units (cfu/ml). persistence shown as survival (%) was obtained by dividing the number of surviving cells following treatment with antibiotics by the total number of cells without treatment. all statistical analyses were performed using origin software (version 9.0). where appropriate, the data were analyzed using student's t test. differences were considered statistically significant at p < 0.05. values in the figure were expressed as the mean of three biological replicates ± one standard deviation. two loci are identified as functional tass belonging to ccdab and hipab putative type ii tass in the ecn chromosome were predicted using a web-based tool ta finder. totally, 16 tass were found (fig. 1a) , which was higher than the average level of tass distribution observed in other e. coli species (~10) (fiedoruk et al. 2015) . among them, most of the ta modules possessed toxins belonging to rele and yhav super-families and antitoxins belonging to hth_xre and prlf super-families, respectively. we chose two of them for further functional analysis in the current study. ecolin_00240 (52351… 52584) and ecolin_00245 (52587…52901), encoding two proteins of 77 aa and 104 aa, were predicted as a typical tas ccdab. the genetic structure analysis showed that they formed the canonical bicistronic structure of the toxinantitoxin system, with the antitoxin gene ecolin_00240 locating upstream of the toxin gene ecolin_00245 with 2 bases in distance (fig. 1b) . these two genes shared a promoter upstream of the ecolin_00240, which was predicted by bprom. additionally, conserved domain analysis of these t w o p r o t e i n s s h o w e d t h a t e c o l i n _ 0 0 2 4 5 a n d ecolin_00240 possessed the domain belonging to ccdb (pfam01845) and ccda (cog5302), respectively, suggesting that they might be a functional tas. similarly, ecolin_08365 (1709929…1711248) and ecolin_08370 (1711248…1711514) were suggested to constitute a functional tas belonging to hipab based on genetic structure and conserved domain analysis. for example, ecolin_08370 located upstream of ecolin_08365 with one base (a) overlapped, and both of them shared a promoter (fig. 1c) . ecolin_08370 (88 aa) and ecolin_08365 (439 aa) possessed the domain included in hipb (prk09726) and hipa (cd17808), respectively. to investigate the activity of individual toxins, the e. coli was used for host killing assay. expression of ccdb induced by iptg significantly inhibited cell growth and viability as shown by a decrease in turbidity and colony-forming units (cfu/ml) ( fig. 1 d and e) . in contrast, the expression of ccda did not affect cell growth and colony-forming activity. besides, the toxic effect of ccdb could be neutralized by co-expressing with ccda. likewise, toxin hipa overexpressed robustly caused cell growth arrest, and this inhibition could be abolished when hipa was co-expressed with antitoxin hipb (fig. 1f) . moreover, toxin hipa remarkably reduced the colony-forming activity of e. coli cells (fig. 1g) . these observations were consistent with previous reports wherein fig. 1 characterization of type ii toxin-antitoxin systems in ecn. a map distribution of putative type ii toxin-antitoxin systems in the ecn chromosome. text in red and black indicates the toxin part and the antitoxin part, respectively. b chromosomal synteny analysis of ecolin_00240 (ccda) and ecolin_00245 (ccdb). c chromosomal synteny analysis of ecolin_08370 (hipb) and ecolin_08365 (hipa). white arrows represent hypothetical proteins. d growth curves of e. coli bl21 (de3) derivatives carrying plasmid peca, pecb, pecab, and petduet-1. e cell growth on lb plate after induction for 4 h. f growth curves of e. coli bl21 (de3) derivatives carrying plasmid peha, pehb, pehab, and petduet-1. g cell growth on lb plate after induction for 4 h. mean values and standard deviations (error bars) are shown from the three biological replicates. red arrows indicate the addition of iptg overexpression of hipa and ccdb resulted in dramatic growth slowdown (germain et al. 2013; gupta et al. 2017) . combined, these results suggested that the ecolin_00240-ecolin_00245 and ecolin_08365-ecolin_08370 of ecn constituted the typical tas ccdab and hipab, respectively. in addition to the canonical hipab described above, a tricomponent hipaab was also observed in the ecn chromosome (fig. 1a) . interestingly, the hipa was split into two genes hipa n (4900381…4900968, encoding n terminus domain) and hipa c (4901039…4901650, encoding c terminus domain). recently, a novel family of tricomponent tas consisted of hips, hipt, and hipb was identified in e. coli wherein the hips and hipt exhibited sequence similarity to n terminus and c terminus of hipa, respectively (vang nielsen et al. 2019) . we further compared the sequence of hipa n and hipa c with that of hips and hipt and found a low similarity between hipa n and hips, as well as hipa c and hipt (data not shown). these suggested that the split hipa and hipb might form a novel tricomponent ta system in ecn, which needs future investigation. ecn shares similar genetic structure of ccdab and hipab with pathogenic e. coli species the amino acid sequences of ccdb, ccda, hipa, and hipb from ecn were used as the reference sequences to search the homologs in other e. coli strains whose genome had been sequenced, including the well-known commensal escherichia coli (cmec) as well as the pathogenic groups such as enterohemorrhagic e. coli (ehec), enteropathogenic e. coli (epec), and enterotoxigenic e. coli (etec) (chaudhuri and henderson 2012) . totally, 79 complete genome sequences were selected (table s2) . we found that ccda was highly conserved in strains belonging to group epec, expec, ehec, and apec since most of them had the homologs that were tightly clustered together with ecn (fig. 2a) . sequence alignment of ccda showed that there was no or only one mutation (s76r) in most of the strains belonging to these four groups while abundant mutations were found in etec and cmec group strains (fig. s3) . similarly, the ccdb was also conserved in pathogenic groups where only two mutations were observed (g10s and e28v) but largely absent in group cmec ( fig. 2b and s4 ). the influence of these mutations on the binding efficiency of ccdab to dna gyrase needs future investigation. in addition to the sequence conservation between ecn and pathogenic e. coli groups, the highly conserved flanking genes adjacent to ccdab operon were also observed, whereas 80% of the selected cmec strains lost the complete ccdab systems (fig. 2c) . the loss of ccdab in cmec might result from the toxin loss during the evolutionary process as usually observed in previous studies (martins et al. 2016; ramisetty and santhosh 2016) . taken together, these findings suggested that ccdab might function similarly in ecn and pathogenic e. coli strains. in comparison to ccdab, the well-known hipab was overall less conserved in different e. coli groups (fig. 3) . for one thing, few homologs were obtained by blast analysis using the ecn hipa and hipb as the reference (fig. 3a and b) . for example, only 8 of the 35 ehec strains had hipa, which resulted from the loss of amino acids (~70 aa) in n terminus (table s2 ). for another, the surrounding genes of hipab were not conserved in e. coli groups since few similar synteny structures were found (fig. 3c) . on the other hand, hipa or hipb of ecn was closely clustered together with that of expec and apec strains, which was consistent with the results obtained in ccdab analysis wherein both ccda and ccdb were more conserved in pathogenic groups. moreover, the highly conserved flanking genes adjacent to hipab operon was found in strains belonging to etec and expec. sequence alignment analysis of hipa and hipb presented that the main mutations in hipa homologs were i104l, d107n, v110i, c112r, k234e, q277r, k290r, k382r, n408t, p435r, r436e, i437y, r438g, y439s/i, and the deletion of k in 440; mutations in hipb were t11m, s53t, a73t, a80s, and a81t ( fig. s5 and s6 ). the influence of these changes on the activity of hipab deserves future study. taken together, the ccdab and hipab from ecn were more prevalent in strains included in groups other than cmec, indicating that despite the probiotic nature of e. coli nissle 1917, it belongs to the phylogenetic group b2, occupied mostly by pathogenic strains responsible for extraintestinal infections (expec). to investigate the physiological role of ccdab and hipab in ecn, crispri-mediated gene knockdown analysis was carried out. in order to inhibit the expression of ccdab and hipab operon, the antitoxin part hipb and toxin part ccdb were selected for the target of dcas9, respectively. the rt-pcr results showed that the expression of ccda was successfully inhibited under the addition of atc since no visible dna fragments were amplified using primer p19 and p20 compared with that of the control group (without atc) (fig. 4a, lanes 2 and 4) . the expression of ccda was restored after removing the inducer atc (fig. 4a, lane 6) , which confirmed that ccda and ccdb were in the same operon and shared a promoter. in order to exclude the experimental bias resulted from the addition of atc, the 16s rdna was used as the endogenous control. as shown in lane 1, lane 3, and lane 5, dna fragments of~250 bp were amplified by using primer p17 and p18 upon either atc was added or not. therefore, the expression of ccdab operon was successfully repressed by targeting the ccdb using crispri. likewise, transcription of hipa was inhibited by targeting hipb since no visible dna fragment was amplified when using primer p21 and p22 (fig. 4b, lane 4) , whereas in the control group (without atc or 16s rdna), a bright dna band was displayed (fig. 4b, lanes 1, 2, 3, and 5 ). this inhibition could be released by removing atc (fig. 4b, lane 6) , indicating that hipa and hipb were in the same operon and shared a promoter. collectively, the expression of ccdab and hipab could be repressed by designing sgrna that targeted either the toxin gene or the antitoxin gene. take the multiple and complex functions of tass into consideration, it is more preferable to investigate several tass simultaneously. to test if the expression of hipab and ccdab could be interrupted simultaneously, a plasmid purcbh carrying sgrna that targeted specifically to hipb the numbers in the bracket such as "4/5" indicate that 4 out of 5 strains in epec have the ccda homologs. c schematic representation of the flanking genes adjacent to ccdab. ehec, enterohemorrhagic e. coli; epec, enteric pathogenic e. coli; etec, enterotoxigenic e. coli; expec, extraintestinal pathogenic e. coli; apec, avian pathogenic e. coli; cmec, commensal e. coli. the strains used in each group were shown in table s2 . the numbers on the left such as "23/35" represent that 23 out of 35 strains in ehec possess a similar synteny structure. arrows with the same color represent the homologous sequences, and white arrows indicate hypothetical proteins. 1, glutathione-regulated potassium-efflux system protein keff; 2, glutathione-regulated potassium-efflux system protein kefc; 3, type 3 dihydrofolate reductase; 4, bis(5′nucleosyl)-tetraphosphatase (symmetrical); 5, co2+/mg2+ efflux protein apag; 6, ribosomal rna small subunit methyltransferase a; 7, 4hydroxythreonine-4-phosphate dehydrogenase; 8, chaperone sura and ccdb was constructed and co-transformed with pdcas9bacteria. after induction, no visible dna fragments of2 50 bp were amplified when using primer p19 and p20 (fig. 4c, lane 5) , and p21 and p22 (lane 6), compared with that of the control group (lane 2 and 3), indicating that the transcription of ccda and hipa were both repressed. this was further supported by the observation that the expression of ccda and hipa were restored upon atc elimination (fig. 4d, lane 2 and 3) . meanwhile, the dna fragments were always obtained when using the cdna reverse transcribed from 16s rrna as the template (fig. 4c, lane 1and 4; fig. 4d, lane 1) . combined, these results suggested that the expression of ccdab and hipab could be inhibited either separately or simultaneously by the crispri system, and crispri was an efficient tool for parallel annotation of multiple tass. the role of ccdab and hipab in biofilm formation was studied. biofilm formation was quantified by the crystal violet assay (sun et al. 2017) . the results showed that the inhibited expression of either ccdab or hipab significantly reduced the biofilm formation of ecn compared with that of control groups (wildtype and non-induced group), with the value of od 540 /od 620 decreased from 1.87 to 1.49, and 1.94 to 1.62, fig. 3 comparative and phylogenetic analysis of hipab among different e. coli serotypes. phylogenetic tree of hipa (a) and hipb (b). c schematic representation of the flanking genes adjacent to hipab. 1, hypothetical protein; 2, trans-aconitate 2-methyltransferase; 3, hypothetical protein; 4, lysr transcriptional regulator; 5, nad (p)-dependent oxidoreductase; 6, fimbrial protein; 7, fimbrial chaperone protein fimc fig. 4 transcriptional repression of ccdab and hipab by crispri. overnight culture of each strain was diluted by 1:100 to 40 ml fresh lb medium supplemented with atc (2 μm) and grown to exponential phase (od 600 = 0.6 ± 0.05). then, cells were collected and used for rna extraction. for restored expression of ccdab or/and hipab, atc was removed by centrifugation and 40 ml fresh lb medium was added to resuspend the cells, following by incubation at 37°c with shaking for another 4 h. then, cells were collected and used for rna and cdna preparation. a verification of the transcriptional silencing of ccdab by rt-pcr. the cdna reverse transcribed from rna prepared from "+" and "−" represent the atc or corresponding primer was added or not, respectively. "+/−" indicates that the atc was initially added and then removed. all primer sequences were displayed in table s1 respectively (fig. 5a) . when the expression of ccdab and hipab were both repressed, the biofilm formation was also sharply decreased with the value dropped from 1.77 to 1.35 (fig. 5a) . beyond the crystal violet staining assay, the in situ morphology of biofilm was also captured by a differential interference microscope (fig. s7) . compared with the control in which no biofilm was observed, the wild-type ecn formed thick biofilm after a 24-h incubation (fig. s7a) , which was consistent with the previous report that ecn could form biofilm inherently (chen et al. 2017) . the silence of ccdab or/ and hipab expression significantly attenuated the biofilm formation and produced an obviously thinned sheet-like biofilm (fig. s7b-d) , which supported the results obtained in the crystal violet assay. collectively, these findings suggested that hipab and ccdab functioned actively in the biofilm formation of ecn. in addition to biofilm formation, the role of ccdab and hipab in the persister formation of ecn was investigated. the persister phenomenon had not been described in ecn, thus to evade the experimental bias, a common e. coli strain mg1655 was used as the control. furthermore, considering that the ecn recombinant strains possessed antibiotic resistance to ampicillin and chloramphenicol or kanamycin, the aminoglycosides gentamycin which kills cells by preventing translation via binding to the ribosome 30s subunit was harnessed (shan et al. 2015) . the mic of gentamycin to ecn, mg1655, and ecn derivatives was 11 μg/ml, 11 μg/ ml, and 9 μg/ml, respectively. in the time-dependent killing assay, the stationary phase cells were exposed to 10-fold the mic of gentamycin over a prolonged time. the cfu/ml of each strain before antibiotic treatment was close (~2 × 10^9). the results showed that persister cells existed in the stationary phase of ecn, depending on the observations that the bulk of the bacterial population was killed rapidly within 4 h and a subsequent surviving population was kept at a frequency around 0.001% (fig. 5b) . the persister fraction of ecn was much lower than that observed in e. coli mg1655 (~0.1%). the silence of ccdab expression significantly decreased the persister frequency of ecn with 3 log units compared with that in wild-type ecn. similarly, knockdown expression of hipab also reduced the formation of persister cells with 2 log units. furthermore, it is noteworthy that when the expression of ccdab and hipab were both repressed, we were unable to isolate viable cells from the culture over the 2-h treatment of gentamycin. taken together, hipab and ccdab were proposed to play an important role in the persister formation of ecn. to investigate the possible targets that were affected by the inhibited expression of ccdab and hipab, nine genes involved in biological processes like dna replication (gyra), sos response (reca and lexa) (durfee et al. 2008) , stringent response (rpos and rela) (durfee et al. 2008) , biofilm formation (foca) (lasaro et al. 2009 ), tass (hipb and ccdb), and posttranscriptional gene regulation (hfq) (henderson et al. 2013) were selected for transcription analysis since these processes were previously reported to be relevant with the stress response of e. coli. first, the transcription of ccdb and hipb were almost blocked when the expression of dcas9 was induced by adding atc (the relative fold change of mutant strain represents that there is a significant difference between two studied groups (*p < 0.05; **p < 0.01; ***p < 0.001) was lower than 0.15 compared with that in wild-type ecn, fig. 6a) . interestingly, the transcription of hipab was significantly inhibited in ecnδccdab, whereas the expression of ccdab was not affected in ecnδhipab, indicating that ccdab might have a positive influence on the expression of hipab. second, when the expression of ccdab and hipab were both repressed, the transcription of gyra and foca were significantly reduced (fig. 6a) . dna and f1c fimbriae are responsible for the biofilm formation of e. coli (lasaro et al. 2009; zhao et al. 2013) ; thus, the results obtained here suggested that hipab and ccdab mediated the biofilm formation of ecn by positively regulating the expression of gyra and foca. third, knockdown expression of either ccdab or hipab resulted in the remarkably increased expression of hfq, reca, rpos, and rela, with the relative fold change ranging from 2.25-to 3.5-fold compared with that in wild-type ecn. moreover, the expression of lexa was downregulated in δccdab mutant while upregulated in δhipab mutant (fig. 6a) . combined, these results suggested that sos response and stringent response mediated by ccdab and hipab contributed to the biofilm formation of ecn. sos response is essential in protecting cells from oxidative stress (bernier et al. 2013; recacha et al. 2019) . thus, it is rational to speculate that the upregulation of these genes will increase the survival of cells upon treatment with antibiotics that are capable of causing oxidative damage. to test our speculation, norfloxacin, a fluoroquinolone, inhibits dna replication via affecting the activity of dna gyrase, was used in the killing assay (gadebusch and shungu 1991) . the norfloxacin mic of ecn wildtype and derivatives was 1 μg/ ml. as expected, when exposed to norfloxacin, wild-type ecn was sharply killed within 6 h (fig. 6b) , whereas the reduction of viable cells in ecnδccdab, ecnδhipab, and ecnδccdabδhipab were relatively slower. moreover, the eventual survival rate of ecn derivatives was significantly higher than that observed in wild-type ecn (p < 0.05), which indicated that knockdown expression of ccdab and hipab induced sos response. conserved distribution of ccdab and hipab shared by the probiotic ecn and pathogenic e. coli mediates non-strictly coincident bioprocesses initially, it was supposed that ecn would contain different tass from those pathogenic e. coli, whereas the contrast results were obtained. almost, the probiotic ecn and pathogenic e. coli, rather than the commensal group, shared the same types and distribution of tass. previous studies revealed that the distribution of tass in e. coli is linked with living niches, and the number of ta loci per strain is different among phylogenetic groups (a, b1, b2, and d) (fiedoruk et al. 2015) . furthermore, horizontal gene transfer is responsible for the tas evolution of e. coli by gene insertion and deletion (ramisetty and santhosh 2016) . therefore, the tass in ecn and other pathogenic strains are suggested to undergo similar evolutionary history, which may result from similar stress stimuli. ccdab and hipab are well-known modules involved in plasmid maintenance and bacterial persistence. as the firstly identified and well-studied tas, plasmid-encoded ccdab is mainly involved in plasmid maintenance via a process known as postsegregational killing (ogura and hiraga 1983) . the chromosomal ccd operon of e. coli o157 is involved in drug tolerance, conferring protection from cell death indicates the same strain. "*" represents that there is a significant difference between two studied groups (*p < 0.05; **p < 0.01; ***p < 0.001) during exposure to multiple antibiotic stress (gupta et al. 2017) . to our knowledge, the role of ccdab in biofilm formation is still unclear to date. our results suggest that ccdab facilitates the biofilm formation of ecn under given conditions (fig. 5a) . accordingly, changes in the transcription level of several genes associated with biofilm formation were observed. the inhibition of ccdab expression induced the upregulated expression of genes involved in the stringent response (rpos and rela) (fig. 6a) , which supported the previous findings in e. coli wherein rpos deletion increased the biofilm formation of strain o157:h7 and a rela mutant of ds291 had improved ability to build biofilm (balzer and mclean 2002; sheldon et al. 2012) . previously, hipa has been restricted to its role in persistence mediation via triggering nutrition starvation through the phosphorylating of glutamyl-trna synthetase (gltx), tryptophanyl-trna synthetase, and other substrates, whereas its role in biofilm formation is rarely reported (germain et al. 2013; semanjski et al. 2018; vang nielsen et al. 2019 ). zhao and co-workers showed that hipa affects biofilm formation through dna release (zhao et al. 2013) . here, transcriptional repression of hipab significantly reduced the expression of gyra, suggesting that one positive role of hipab in regulating the biofilm formation of ecn is achieved by increasing the dna synthesis. knockdown expression of hipab also caused the upregulated expression of genes involved in the stringent response, which further suggests that stringent response may function negatively in the biofilm formation of ecn. interestingly, the expression of hfq and reca were upregulated when the transcription of hipab and ccdab were inhibited, contradicting the previous finding that reca deletion reduced the biofilm production by 22-80% in e. coli when exposed to the sub-mic of ciprofloxacin (recacha et al. 2019 ). our results suggest that reca may act negatively in the biofilm formation of ecn. thus, how sos response contributes to the biofilm formation of ecn needs future investigation. rna chaperone hfq is a well-known stress response regulator and functions primarily in srna-mediated post-transcriptional gene regulation via facilitating the pairing between srnas and their target mrnas (henderson et al. 2013; liu et al. 2019 ). e. coli seems less prone to form biofilm upon the deletion of hfq, which contradicts our observation here that upregulation of hfq expression corresponded to lower biofilm formation (kulesus et al. 2008; ramos et al. 2014) . furthermore, it is interesting that decreased expression of foca that is responsible for f1c fimbriae synthesis in ecn only appeared in case of the simultaneous knockdown expression of ccdab and hipab (fig. 6a ). f1c fimbriae has been shown to be indispensable in the biofilm formation of e. coli wherein foca mutant is defective in biofilm building (lasaro et al. 2009 ). thus, we propose that the contribution of f1c fimbriae to the biofilm formation of ecn is mediated by ccdab and hipab on multiple levels. overall, the mechanisms involved in the biofilm formation of ecn are complicated; ccdab and hipab are suggested to achieve a collectively positive regulating role at least (fig. 7) . studies of bacterial persistence in e. coli have been widely performed, of which toxin-antitoxin systems are generally accepted to be the regulators of persistence (page and peti 2016; semanjski et al. 2018) . however, t h e s e f i n d i n g s h a v e r e c e n t l y b e e n c h a l l e n g e d (goormaghtigh et al. 2018 ). nevertheless, we suggest here that the persister formation of ecn in the stationary phase is mediated by ccdab and hipab. on the other hand, it was interesting to find that knockdown expression of hipab induced upregulation of rpos and rela expression, which was usually observed in case of hipa overexpression (durfee et al. 2008; kaspy et al. 2013 ). this may be explained that the stringent response is simultaneously controlled by other mechanisms while the silence of hipab expression seems to promote this process. besides, we found that the transcriptional halt of hipab induced sos response (fig. 6b) . the direct link between hipab and sos response is rarely reported in e. coli, whereas our results suggest that persister formation mediated by hipab may have an indirect correlation with sos response in ecn (fig. 7) . ccdb inhibits dna replication by binding to gyrase-dna complex and blocking the dna polymerase passage, leading to dna doublestrand breaks and cell growth inhibition (bahassi et al. 1999 ). thus, it is possible that the repressed expression of ccdb will promote dna replication. this case was found in ecn where knockdown expression of ccdab resulted in the upregulation of gyra expression. moreover, it was surprising to find the upregulation of reca expression in δccdab mutant since this generally happens upon ccdb overexpression (tripathi et al. 2012 ). this could not be ascribed to the experimental bias since the survival of the δccdab mutant was significantly increased during exposure to norfloxacin (fig. 6b) , indicating that expression of reca is regulated by other unknown mechanisms. thus, a future study aiming to explore the molecular mechanisms underlying intrinsic correlation between tass (e.g., ccdab), sos response, and persister formation will enhance our understanding of the persistence phenomenon in ecn. toxin-antitoxin systems, the potential regulator in ecn for bacterial interference? the use of antimicrobial compounds in the treatment of bowel disorders caused by the infection of pathogenic e. coli and other fatal bacteria is controversial because of the emergence of antibiotic resistance (persistence) as well as observed increased endotoxin release after antibiotic exposure in some enteropathogen (bielaszewska et al. 2012; mohsin et al. 2015) . in past decades, the probiotic ecn has been widely used in the treatment of intestinal disorders such as ulcerative colitis, crohn's disease, and inflammatory bowel disease due to its high ability in competing with enteropathogen (chen et al. 2017; huebner et al. 2011; rembacken et al. 1999) . ecn has been reported to have a better capacity in biofilm formation and help to outcompete the most known pathogenic e. coli (hancock et al. 2010) . however, in addition to the finding that f1c fimbriae are necessary for the biofilm formation of ecn, largely unknown factors involved in the regulation of biofilm formation are waiting for elucidation (lasaro et al. 2009 ). besides, whether the formation of persister cells helps ecn combat with other bacteria and survive under hostile environments in the intestine remains elusive. the results obtained in this study shed some light on this field. thus, to prompt the application of ecn in bacterial interference, a future study focusing on the elucidation of the correlation between tass, robust biofilm, and persister formation of ecn during intestine colonization will be helpful. crispri is proposed to be an efficient tool in annotating toxin-antitoxin systems in situ a common difficulty encountered during the study of specific tas is functional redundancy with other tass as well as the number duplicates (ramage et al. 2009 ). deletion and restoration of gene expression are the methods that have been usually adopted to annotate the function of a given gene. in contrast to one gene, sometimes, multiple tass needed to be knocked out before there is an observable phenotype, which asks for a long period for molecular manipulation (kim et al. 2009; kolodkin-gal et al. 2009 ). in this case, an efficient and convenient molecular tool is of great importance. currently, the crispr interference (crispri) system has been widely employed in the regulation of gene expression (kim et al. 2020; qi et al. 2013; wang et al. 2019) . however, to the best of our knowledge, it is the first time that the crispri tool is applied in the functional fig. 7 model of identified hipab and ccdab-mediated genes in ecn. transcriptional silencing of hipab by crispri inhibits the expression of a gene involved in dna replication (gyra) and upregulates the expression of genes involved in stringent response (rpos and rela), sos response (reca and lexa), and rna interaction (hfq); transcriptional silencing of ccdab by crispri inhibits lexa expression and upregulates the expression of gyra, rpos, rela, reca, and hfq. these changes eventually lead to the reduced biofilm formation and persister formation upon gentamycin treatment in ecn annotation of type ii tass. to inhibit the expression of either ccdb or hipa, we designed three n20 sequences for each gene. the results showed that each of them was functional but with different efficiency (data not shown). by choosing the best n20 sequences, two chromosomal tass ccdab and hipab in ecn were annotated smoothly, suggesting that crispri could be a strong candidate tool in tas study among e. coli as well as other bacteria. on the other hand, crispri is capable of producing different levels of the expression of a targeted gene either by knocking down or activating, thus helping to study the behavioral changes of cells upon various conditions (tao et al. 2018) . this makes sense when we consider the clinical application of ecn where cell viability is one important factor that determines the therapeutic process. in general, we could enhance the oral delivery to guarantee the therapeutic effect. our findings suggest that sustaining the cell viability of ecn dynamically under the intestine environment by regulating the expression of tas is preferable and constructive. in addition, genetic engineering of the ecn chromosome usually happens for ecn-based vaccine development, in which the increased or decreased gene expression of interest is necessary (chaudhari et al. 2017; ou et al. 2016) . in this case, crispri would be a robust tool for the regulation of gene expression in ecn. in conclusion, we reasoned that the ecolin_00240-ecolin_00245 and ecolin_08365-ecolin_08370 operon of ecn encoded ccdab and hipab, respectively. by crispri, ccdab and hipab were shown to be involved in the formation of biofilm and persister cells in ecn. moreover, we evidenced that biofilm formation and cell persistence regulated by ccdab and hipab were associated with the sos and stringent response in ecn (fig. 7) , which may be helpful for the future study of the intestinal colonization of ecn. furthermore, the crispri approach is proposed to be an efficient and precise tool in deciphering multiple tass. bile enhances cell surface hydrophobicity and biofilm formation of bifidobacteria determination of minimum inhibitory concentrations interactions of ccdb with dna gyrase. inactivation of gyra, poisoning of the 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gene expression control in yersinia pestis by using an optimized crispr interference system the bacterial antitoxin hipb establishes a ternary complex with operator dna and phosphorylated toxin hipa to regulate bacterial persistence characterization and comparative analysis of toxin-antitoxin systems in acetobacter pasteurianus toxin-antitoxin systems and their role in disseminating and maintaining antimicrobial resistance construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in bacillus subtilis escherichia coli toxin gene hipa affects biofilm formation and dna release publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions j.x. conducted lab work and data analysis. k.x. interpreted the data, designed the experiments, and drafted the manuscript. c.q. and p.l. conducted part of the lab work and data analysis. y.l. contributed to the bioinformatics analysis and paper review. x.l. conceived of the study, performed data review, and contributed to the paper writing. all authors read and approved the final manuscript. the work was financially supported by grants from the natural science foundation of zhejiang province (ly19c200002) to xinle liang. this paper does not contain any studies with human participants or animals. the authors declare that they have no conflicts of interest. key: cord-288673-ku3tmjd3 authors: sabotič, jerica; kos, janko title: microbial and fungal protease inhibitors—current and potential applications date: 2012-01-05 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3834-x sha: doc_id: 288673 cord_uid: ku3tmjd3 proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. because of their essential roles, their proteolytic activity needs to be tightly regulated. therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. in medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. they can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. they are also valuable tools for simple and effective purification of proteases, using affinity chromatography. because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well. applications of protease inhibitors are intimately connected to the proteases they inhibit. so, as a preface to protease inhibitors, an overview of proteases with the modes of regulation of their proteolytic activity is provided. then, known microbial and fungal protease inhibitors are reviewed, with the emphasis on protein (tables 1 and 2 ) rather than small-molecule protease inhibitors (table 3) . finally, their potential applications in the fields of medicine, crop protection and biotechnology are described, based on their target proteases. microorganisms (prokaryotes, yeasts and filamentous fungi) and higher fungi or mushrooms have been selected for review since protease inhibitors of microbial origin have already proven useful in many different applications. higher fungi have emerged as a valuable source of new protease inhibitors with unique characteristics only in the last decade and therefore offer great potential for future applications. proteases, also called peptidases or proteolytic enzymes, constitute a large group of enzymes that catalyse the hydrolysis of peptide bonds. cleavage of peptide bonds can be general, leading to complete degradation of protein substrates into their constituent amino acids, or it can be specific, leading to selective protein cleavage for post-translational modification and processing. peptidases that cleave peptide bonds at the termini of polypeptide chains are called exopeptidases, while endopeptidases cleave peptide bonds within the polypeptide chain. peptidases are classified according to their catalytic type into aspartic, cysteine, glutamic, serine and threonine peptidases, according to the main, functional amino acid residue at the active site. metallopeptidases, on the other hand, are those whose catalytic activity depends on the presence of a divalent metal ion bound within the active site. in the mer-ops database (http://merops.sanger.ac.uk/), peptidases are classified further into families, according to their sequence similarity, and into clans, according to their structural similarity. there are 226 peptidase families assigned in the merops database (release 9.5, july 2011) and 57 clans, based on structural data (barrett 2001; rawlings et al. 2010) . peptidases are present in all living organisms, including viruses, bacteria, archaea, protists, fungi, plants and animals. serine peptidases form the most abundant class, followed by metallo-, cysteine, aspartic and threonine peptidases. there has been an explosive growth of the number of peptidase families observed in eukaryotic organisms, there being 100 peptidases in bacterial genomes and half as many in archaeal genomes and from 400 to 700 peptidase genes in plant and mammal genomes. furthermore, there is a striking difference between the compositions of eubacterial and eukaryotic degradomes, (i.e. the complete set of proteases present in an organism). sixteen peptidase families constitute the core of the nearly ubiquitous peptidase families present in all living forms. additional 34 peptidase families are widely distributed in eukaryotic organisms, while another ten are unique to higher metazoan organisms, performing mainly limited proteolysis in extracellular environments (page and di cera 2008; rawlings et al. 2010) . in addition to the merops database, information on proteases can be found in several other online databases, including the degradome database (http:// degradome.uniovi.es/) (quesada et al. 2009 ) and the proteolysis map (pmap) (http://www.proteolysis.org/) that comprises five α 2 m a1a, a2a, c1a, c2a, c11, m4, m10a, m10b, m12a, m12b, s1a, s1b, s8a a underlined families include protease inhibitors exclusively of microbial and/or fungal origin b × denotes the number of sequence homologues found in each group of organisms: × less than 10, ×× 11-200, ××× 201-1000 and ×××× more than 1000 (armstrong and quigley 1999; sottrup-jensen 1989) serine protease inhibitors ovomucoid (kazal-type) chymotrypsin (s1) and subtilisin (s8) families tight-binding, laskowski described in stramenopiles oomycetes (fungus-like microorganisms distantly related to fungi); e.g. involved in pathogenicity of phytophtora infestans (tian et al. 2004; rawlings and barrett 2011) aprotinin i2 chymotrypsin family (s1) broad inhibitory specificity (ascenzi et al. 2003; rawlings and barrett 2011) peptidase b inhibitor i9 subtilisin family (s8) bacterial inhibitors are propeptides of subtilisin-like proteases. fungal inhibitors are separate polypeptides, e.g. pleurotus ostreatus proteinase a inhibitor 1 poia1 and yeast proteinase inhibitor 2 yib2 they are potent but unstable inhibitors, gradually degraded by subtilisin (ascenzi et al. 2003; dohmae et al. 1995; kojima et al. 1999; kojima et al. 2005; kojima et al. 1997; rawlings and barrett 2011) marinostatin i10 proteases of family s8 (subtilisin) and certain proteases of family s1 (chymotrypsin) tight-binding, laskowski. structure stabilized by two internal ester bonds that are essential for their inhibitory activity exclusive to marine bacteria (kanaori et al. 2005; rawlings and barrett 2011) ecotin i11 chymotrypsin family (s1) tight-binding, laskowski for primary binding site. active as dimers, each monomer binds the protease at two binding sites ecotins from enterobacteria and parasites perform a protective role against host digestive proteases and target host proteases to facilitate colonization. structure enables inhibition of multiple proteases with the chymotrypsin fold (eggers et al. 2001; eschenlauer et al. 2009; rawlings and barrett 2011) streptomyces subtilisin inhibitor (ssi) family s8 (subtilisin, kexin), family s1 (trypsin, plasmin) and the metalloprotease griselysin (family m4) tight-binding, laskowski exclusive to bacterial actinomycetales order. they probably control endogenous proteases involved in proteolytic activation of transglutaminase (kantyka et al. 2010; taguchi et al. 1997; tsuyuki et al. 1991; rawlings and barrett 2011) carboxypeptidase y inhibitor i51 serine carboxypeptidase y (family s10) a defensive role against predatory insects has been shown, but a regulatory endogenous role is also possible (avanzo et al. 2009; odani et al. 1999; sabotič et al. 2012; rawlings and barrett 2011) human elastase-2 and endogenous aspergillus elastases (family s1) unknown homologues have been found in a few other ascomycete species and in proteobacteria, but none was characterized biochemically (okumura et al. 2008; okumura et al. 2006; rawlings and barrett 2011) serpin i4 chymotrypsin (s1) and subtilisin (s8) families. some members inhibit also cysteine proteases of papain (c1) and caspase (c14) families trapping. suicide inhibitors in which a rapid conformational change traps the cognate protease in a covalent complex the physiological role of microbial serpins has been proposed to be to protect the cellulosedegrading apparatus (cellulosome) against proteolytic degradation. the only fungal serpin (celpin), was characterized from the anaerobic fungus piromyces sp. e2 (kantyka et al. 2010; law et al. 2006; roberts et al. 2004; steenbakkers et al. 2008) cysteine protease inhibitors thyropin i31 papain-like proteases (family c1), and equistatin inhibitor unit 2 inhibits an aspartic protease cathepsin d (family a1) tight-binding present in animals and in one bacterial pathogen (coxiella burnetii) that has presumably acquired the gene by lateral transfer (kantyka et al. 2010; rawlings and barrett 2011) survivin i32 caspases-aspartate-specific cysteine proteases (family c14) tight-binding, several mechanisms survivin plays a dual role as a mitotic regulator of cell division and as an inhibitor of caspase activation in the process of apoptosis. fungal homologues have been identified in ascomycete and a few basidiomycete genomes. the fission yeast homologue is a conserved chromosomal passenger protein (huang et al. 2005; luthringer et al. 2010; rawlings 2010) chagasin i42 protozoan and mammalian papain-like cysteine proteases (family c1) tight-binding parasitic chagasins are involved in regulating endogenous cysteine proteases essential for their life cycle. in bacteria and archaea, chagasins serve as endogenous regulators, and in some pathogenic species, they also serve a protective role against host proteases (kantyka et al. 2010; santos et al. 2006) clitocypin (i48) and macrocypin (i85); together named mycocypins i48, i85 papain-like cysteine proteases (family c1) and legumain (family c13) and serine protease trypsin (family s1) tight-binding. mycocypins are small and exceptionally stable proteins. they have a β-trefoil fold formed by the core six-stranded β-barrel that supports 11 loops which provide a versatile surface for the inhibition of several types of proteases unique to basidiomycetes. they probably have an endogenous regulatory role or a role in defence against pathogen infection and/or predation by pests. a defensive role for mycocypins is further supported by their high genetic variability and conformational stability as well as a broad inhibitory profile (brzin et al. 2000; kidrič et al. 2002; renko et al. 2010; sabotič et al. 2007a; sabotič et al. 2006; sabotič et al. 2009b the high specificity is the result of the structural stabilization of the ia3 inhibitor in complex with saccharopepsin since the unstructured inhibitor in solution forms an alpha helix upon interaction with the enzyme active site (green et al. 2004; phylip et al. 2001) different databases (cutdb, pathwaydb, proteasedb, substratedb and profiledb) (igarashi et al. 2009 ). the occurrence of proteases in all living organisms indicates their critical role in essential metabolic and regulatory functions in many biological processes. proteases are important in the production of nutrients for growth and proliferation. extracellular proteases catalyse the hydrolysis of proteins into smaller peptides and amino acids for subsequent absorption into cells, constituting a very important step in nitrogen metabolism. proteases perform critical regulatory functions in numerous physiological processes since they regulate the fate, localization and activity of many proteins, modulate protein-protein interactions and contribute to the generation, transduction and amplification of molecular signals. proteases are involved in a wide span of cellular and metabolic processes, including regulation of gene expression, dna replication, transport of proteins, cell growth and differentiation, cell cycle, heat shock response, sos response to dna damage, misfolded protein response, oxidative stress response and programmed cell death (lopez-otin and bond 2008; rao et al. 1998 ). furthermore, in plants, proteases are important in the build-up and breakdown of seed storage proteins during seed germination, protein remobilization upon organ senescence and in many developmental processes such as embryogenesis, chloroplast biogenesis, photomorphogenesis, hormone signalling, flower development, pollen-pistil interaction and local and systemic defence responses against pathogens and herbivores (simoes and faro 2004; salas et al. 2008; schaller 2004; van der hoorn 2008) . moreover, in animals, proteases are involved in tissue morphogenesis and remodelling, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, haemostasis, blood coagulation, inflammation, immunity, autophagy and senescence (lopez-otin and bond 2008). proteases of microbial and fungal origin offer a wide range of biotechnological applications. alkaline proteases have been used in the detergent industry for over 50 years. proteases with elastolytic and keratinolytic activities have been used in the leather industry for de-hairing and baiting of skins and hides. the food industry uses proteases in cheese making, baking, preparation of various protein hydrolysates, meat tenderization and manufacturing protein-rich diets. in the pharmaceutical industry, proteases have found uses as therapeutic agents as well as additives in preparations of slow-release dosage forms. bioprocessing of used x-ray films for silver recovery involves the use of alkaline proteases. proteases allow potential applications for the management of wastes from various food processing industries and from household activities. in addition to industrial and medical applications, proteases are used in basic research; for example, proteases with very selective peptide bond cleavage are used in protein sequencing, unselective proteinase k is used in nucleic acid isolation, and trypsin is widely used in maintaining animal cell cultures (kumar and takagi 1999; rao et al. 1998) . there is also the downside to proteases as some are important virulence factors of many pathogenic bacteria, parasites and viruses. these proteases are involved in acquiring nutrients for growth and proliferation through host tissue degradation and evasion of host immune defences. in addition to colonizing and facilitating dissemination functions, they are also involved in evading the host immune system by interrupting the cascade pathways, disrupting the cytokine network, excising cell surface receptors and inactivating host protease inhibitors (maeda 1996; travis and potempa 2000; supuran et al. 2002) . because proteases play essential roles in life and death processes in all living organisms and because peptide bond hydrolysis is irreversible, anomalies in proteolytic activities lead to numerous pathological conditions, including cancer, neurodegenerative disorders and inflammatory and cardiovascular diseases, as well as bacterial, viral and parasitic diseases (lopez-otin and bond 2008; turk 2006) . activity of proteases is regulated on several levels, including regulation of gene expression at transcriptional and post-transcriptional levels, synthesis as inactive zymogens, blockade by endogenous inhibitory proteins, spatial and temporal compartmentalization, post-translational modification (glycosylation, phosphorylation, co-factor binding), proteolysis and degradation (lopez-otin and bond 2008). protein protease inhibitors constitute a very important mechanism for regulating proteolytic activity. they can be classified approximately according to the class of proteases they inhibit (for example, serine or cysteine protease inhibitors). however, those composed of multiple inhibitor units and pan-inhibitors (such as α 2 -macroglobulin of family i39) that target proteases of different catalytic classes prevent unambiguous classification. a more detailed classification is included in the merops database (http://merops.sanger. ac.uk/), which follows a hierarchy similar to that for proteases. protease inhibitors are grouped into families based on sequence homology and into clans based on protein tertiary structure. in release 9.5 (4 july 2011) of the mer-ops database, there are 71 families of protease inhibitors, and those with available three-dimensional structural data have been assigned to 39 different clans (rawlings 2010) . of the 71 families, 27 include members of microbial and fungal origin (tables 1 and 2 ). of these, seven families include members of exclusively bacterial origin (i10, i16, i36, i38, i57, i58, i69), and five families include members of exclusively fungal origin (i34, i48, i66, i79, i85). in addition to protein protease inhibitors, the merops database includes a list of small-molecule inhibitors that are well known and widely used. many of them have been synthesized in the laboratory; however, those that occur naturally (table 3 ) have been isolated from bacteria and fungi (rawlings 2010) . there are two general mechanisms of protease inhibition, namely, irreversible "trapping" reactions and reversible tight-binding reactions. trapping reactions work only on endopeptidases and are the result of a conformational change of the inhibitor triggered by cleavage of an internal peptide bond by the host protease ( fig. 1a ). only three families utilize a trapping mechanism: i4 (serpins), i39 (α 2 -macroglobulin) and i50 (viral caspase inhibitors). reversible tight-binding inhibition is widespread, the best known being the "standard canonical" or "laskowski mechanism", in which the inhibitor has a peptide bond that is cleaved by the peptidase active site in a substrate-like manner. the inhibitor is only slowly released due to the conformational stability of the stabilized loop that can mimic a substrate. this mechanism has been conclusively demonstrated only for inhibitors of serine proteases. other reversible tight-binding protease inhibitors physically block the protease active site by high-affinity binding to sites on either side of the active site (fig. 1b, c) . binding of an inhibitor to the active site can also be irreversible, when an electrophilic reactive group of the inhibitor forms a covalent bond with an amino acid residue in the enzyme active site. there are also some inhibitors that block the exosites, to which substrate binding occurs in addition to the active site in some proteases (christeller 2005; rawlings 2010; ). the families of protein protease inhibitors that include members of microbial and fungal origin are described in table 2 , and information of their distribution in taxonomic groups is given in table 1 . protease inhibitors are described in groups according to the catalytic class of protease they inhibit and following the merops inhibitor classification (rawlings 2010; rawlings and barrett 2011) . since prokaryote-derived protease inhibitors have been reviewed recently (kantyka et al. 2010 ), more information on protease inhibitors of fungal origin, including yeasts, filamentous fungi and mushrooms, is provided here. among the small-molecule protease inhibitors isolated from bacteria and fungi (table 3) , there are several that show broad inhibitory specificity and inhibit proteases of different catalytic classes. several inhibit both serine and cysteine proteases (antipain, chymostatin, leupeptin), serine and metalloproteases (bestatin, puromycin), metallo-and cysteine proteases (amastatin) or metallo-, cysteine and serine proteases (bacitracin a). of the small-molecule cysteine protease inhibitors, the best known is e-64 (1-[l-n-(trans-epoxysuccinyl) leucyl] amino-4-guanidinobutane), an irreversible inhibitor originally isolated from aspergillus japonicus (hanada et al. 1978) and routinely used as a class-specific cysteine protease inhibitor. a number of e-64 analogues have been synthesized in order to improve selectivity for a particular cysteine protease. several inhibitors are specific for metalloproteases and inhibit more than one protease family (e.g. phosphoramidon). the only natural small-molecule inhibitor of aspartic proteases is pepstatin, originally isolated from various species of actinomycetes (umezawa et al. 1970) , which inhibits several families of aspartic proteases. it is a hexa-peptide containing the unusual amino acid statine (rawlings 2010) . proteases play an important part in almost every biological process; therefore, unregulated activity often leads to disease. in this review, only excessive proteolysis will be addressed as it is the one that can be reversed by protease inhibitors. excessive proteolysis plays an important role in cancer and in cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. due to the obvious relevance of protease inhibitors, they have been studied extensively with the intent to develop therapeutic drugs (drag and salvesen 2010; turk 2006; haq et al. 2010) . proteases that have a potential as therapeutic targets are reviewed, according to their catalytic type, for each group of disease-causing organisms and for other human diseases. information on the availability of protease inhibitors for each protease described is provided, with the emphasis on those for which specific inhibitors have not yet been identified. proteolytic processing of virus polyprotein into structural and non-structural proteins is an essential part of the viral replication cycle, making the proteases an important antiviral drug target. several viral proteases have been studied as therapeutic targets. although proteases of any virus could be potential antiviral targets, viruses that cause chronic diseases (e.g. hiv, herpes virus) and those that could cause large-scale epidemics (e.g. sars coronavirus, dengue virus) have received most attention. several protease inhibitors acting against the human immunodeficiency virus 1 (hiv-1) protease, a homodimeric aspartic protease, have been used in treating hiv-1 infection. fig. 1 examples of protease inhibitors utilizing irreversible "trapping" reaction (a) and reversible tight-binding reactions (b and c). proteases are shown in light grey, their active site residues in black and inhibitors in dark grey. a serine protease trypsin in complex with serpin (family i39) (pdb id 1k9o). the protease cleaves the reactive centre loop of serpin, which triggers a conformational change in the inhibitor and trapping of the protease in an inactive covalent complex. b cysteine protease cathepsin v in complex with clitocypin (family i48) (pdb id 3h6s). the inhibitor binds to the protease active site cleft and obstructs access of substrate. c aspartic protease plasmepsin iv in complex with the small-molecule inhibitor pepstatin a (pdb id 1ls5). the inhibitor binds in the active site of the protease these are all low molecular weight peptidomimetic inhibitors whose design has been based on the structures of the compounds binding to the protease active site. in therapy, they are usually used in combination with inhibitors of reverse transcriptase (abbenante and fairlie 2005; anderson et al. 2009 ). in addition to a number of designed synthetic inhibitors, a potent peptidic inhibitor of hiv-1 protease of bacterial origin (atbi) has been found in an extremophilic bacillus sp. (dash and rao 2001; vathipadiekal et al. 2010) . other targeted viral proteases belong to the serine catalytic type. human cytomegalovirus (hcmv) is one of eight human herpes viruses and is widespread in populations worldwide, with infection rates of 80-100%. it causes asymptomatic infections in healthy individuals but high morbidity and mortality in immunocompromised individuals. a few inhibitors of the cytomegalovirus protease have been described from bacterial (streptomyces) and fungal (cytonaema) origins (stoeva and efferth 2008; anderson et al. 2009 ). an additional target in antiviral therapy against cytomegalovirus is the proteasome, where the aim is to hinder the recruitment of host proteins by the virus for its replication. several synthetic and a few natural proteasome inhibitors (e.g. lactacystin from streptomyces lactacystinaeus) are known and have been reported to obstruct replication of several viruses, including influenza virus, herpes simplex virus type 1, paramyxovirus and rhabdoviruses, as well as cytomegalovirus (kaspari and bogner 2009 ). the serine proteases ns3 and ns2 of flaviviruses are targets for antiviral drug development against hepatitis c virus and dengue virus, with the former blood-transmitted virus causing various liver diseases (including cirrhosis and liver cancer) and the latter being a mosquitotransmitted disease causing dengue hemorrhagic fever. several structure-based designed low molecular weight inhibitors are in different phases of clinical evaluation (anderson et al. 2009; lange et al. 2010; tomlinson et al. 2009 ). the coronavirus associated with severe acute respiratory syndrome, sars, encodes a chymotrypsin-like cysteine protease m pro that is similar to picornavirus 3c protease. since the 2003 sars global outbreak, several strategies of structure-based design of low molecular weight protease inhibitors have been applied in the search for antiviral drugs against sars (anderson et al. 2009; sirois et al. 2007 ). the first to be considered were the protease inhibitors targeting the picornavirus 3c protease. the picornaviruses, which encode a 3c protease, are important human and animal pathogens such as poliovirus, hepatitis a virus, coxsackievirus, human rhinovirus and foot-and-mouth disease virus. inhibitors targeting the 3c protease of human rhinoviruses that cause common cold, as well as 3c proteases of other picornaviruses and coronaviruses, have been developed based on structural data, but none has yet successfully passed all the phases of clinical evaluation (neubauer et al. 2009; wang and liang 2010) . due to the rapid development of resistance in viruses, the search for novel strategies for developing inhibitors targeting different sites on proteases is encouraged, including the search for novel lead compounds from natural sources and structure-based drug development. bacterial pathogens employ an array of virulence factors that enable their colonization, evasion of host defences and dissemination. proteases are important virulence factors of many pathogenic bacteria, which play roles in acquiring nutrients by direct degradation of host tissue components. the even more important aim of disrupting host immune response and signalling cascades has been reviewed by potempa and pike (2009) . most currently available antibiotics target bacterial cell wall synthesis or protein synthesis. in the light of rapidly spreading antibiotic resistance, bacterial proteases are promising targets for the design of novel antibiotics. metalloproteases are most abundantly represented in primary and opportunistic pathogens, although all catalytic classes are found. these proteases are often associated with mobile genetic elements (plasmids, pathogenicity islands, integrated phages), and their expression is not constitutive but regulated through environmental or cellular signals (travis and potempa 2000; wladyka and pustelny 2008) . omptins (outer membrane proteins t) are aspartic proteases (family a26) found in several gram-negative bacteria, including the pathogenic species escherichia coli (ompt), yersinia pestis (pla), shigella flexneri, shigella dysenteriae (sopa), salmonella enterica (pgte), legionella pneumophila (lpa) and plant pathogens agrobacterium tumefaciens and erwinia pyrifoliae (plaa). omptins are bacterial virulence factors and, in addition to their proteolytic activity, possess adhesive and invasive activities. they modulate the coagulation system since they act as plasminogen activators (ompt, pla, pgte, lpa), inactivate tissue factor pathway inhibitor (ompt, pla, pgte), degrade thrombin-activatable fibrinolysis inhibitor (pla, pgte), degrade the complement system proteins (pla, pgte) and antimicrobial peptides (ompt, pla, pgte), and process autotransporters (ompt, sopa). lipopolysaccharide (lps) is required for their enzymatic activity. omptins have a unique catalytic mechanism that combines the elements of both serine and aspartic proteases, and partial inhibition by serine protease inhibitors has been reported (hritonenko and stathopoulos 2007; valls seron et al. 2010; yun et al. 2009 ). other than a weak substrate-based peptide inhibitor with a d-arg, shown to inhibit ompt (dekker et al. 2001) , and a colicin immunity protein shown to protect colicin e2 from degradation by ompt in escherichia coli (duche et al. 2009) , no specific inhibitors have been reported. no specific inhibitor has so far been found for the exfoliative toxin a, a glutamate-specific serine protease (family s1) produced by staphylococcus aureus, which is the causative agent in staphylococcal scalded skin syndrome. the target of the toxin is a transmembrane glycoprotein desmoglein-1 of the cadherin superfamily, which plays an important role in keratinocyte cell-cell adhesion (ladhani 2003) . immunoglobulin a1 proteases (iga1 proteases) are serine proteases (family s6) produced by several pathogenic bacteria, including species causing bacterial meningitis, haemophilus influenzae, neisseria meningitidis and streptococcus pneumoniae. the iga1 proteases enable colonization of human mucosal surfaces by cleaving the secretory iga antibodies, thus disrupting the specific immunity response. except for an early report on substrate analogue inhibitors (burton et al. 1988 ) and small synthetic peptide inhibitors (bachovchin et al. 1990 ) of neisseria gonorrhoaeae iga1 protease, there has been no further development of iga1 protease inhibitors (mistry and stockley 2006) . other immunomodulating serine proteases are c5a peptidases from streptococci (family s8)-those of group a (streptococcus pyogenes scpa) and group b (streptococcus agalaciae scpb) streptococci have been described in more detail. streptococcus pyogenes is the causative agent of pharyngitis and also causes rheumatic fever and skin infections, which can develop severe complications, including toxic shock syndrome. c5a peptidases are important for streptococcal pathogenesis as they specifically cleave complement c5a and therefore prevent the recruitment of phagocytic cells to the infection site (cheng et al. 2002; collin and olsen 2003) . antibodies raised against c5a peptidase were used to inhibit c5a peptidase in vivo (park and cleary 2005) , but no peptidase inhibitors have been described. serine proteases are important pathogenesis factors in bacteria involved in dental diseases. treponema denticola is a spirochete implicated in the progression of periodontal diseases. a serine protease, trepolisin (also called dentilisin), of family s8 is an important pathogenesis factor, a mediator of cytopathic effects by degrading host proteins, including extracellular matrix components and host protease inhibitors (sela 2001) . the broad-range inhibitor of serine proteases (families s1 and s8), chymostatin, inhibits trepolisin; however, no specific inhibitors have been described. another important oral cavity pathogen involved in periodontal disease, porphyromonas gingivalis, in addition to a few cysteine proteases (discussed further in the following), produces a serine protease, a prolyl tripeptidyl peptidase ptpa (family s9), which is involved in degrading host connective tissue, providing nutrients for bacterial growth (banbula et al. 1999) . a substrate-based specific inhibitor of ptpa has been developed (xu et al. 2008 ). bacterial type i signal peptidases spase (family s26) are serine proteases widespread among gram-negative and gram-positive bacteria and are membrane proteases required for processing newly synthesized secreted proteins. in addition to their essential role in bacterial viability, they are important antimicrobial drug targets as they are involved in the secretion of many virulence factors (paetzel et al. 2000) . synthetic penem inhibitors have been developed for inhibiting both gram-negative (escherichia coli) and grampositive (staphylococcus aureus, staphylococcus epidermidis) spases. the various penem derivatives display different degrees of activity against these pathogenic bacteria spases (allsop et al. 1995; harris et al. 2009 ). recently, substratebased peptide aldehydes have been shown to be promising inhibitors of escherichia coli and staphylococcus aureus spases (buzder-lantos et al. 2009 ). however, the in vitro inhibitory activity and in vivo antimicrobial activities of these inhibitors did not correlate well, so further optimization of and search for novel spase i inhibitors are expected. no specific inhibitors have been described for the streptococcal pyrogenic exotoxin b (speb, streptopain), a cysteine protease (family c10) produced by all strains of streptococcus pyogenes. it is a multifunctional protease and an important pathogenesis factor with several immunomodulating activities, including release of proinflammatory molecules, degradation of extracellular matrix, cleavage of igg in the hinge region and degradation of other immunoglobulins. in addition to class-specific cysteine protease inhibitors, a peptide derivative was shown to inhibit speb, as well as α 2 -macroglobulin and an s-nitrosylated form of α 1 -protease inhibitor (collin and olsen 2003) . ides (family c66) is another cysteine protease from streptococcus pyogenes that specifically cleaves igg, its only known substrate (vincents et al. 2004 ). in addition to specific, inhibitory igg antibodies (akesson et al. 2006) , synthetic reversible inhibitors were designed, with aldehyde compounds being the most promising; however, no specificity data are yet available for these inhibitors (berggren et al. 2009 ). staphylococcus aureus causes a range of diseases, from mild skin infections to life-threatening disorders, including septicaemia, endocarditis, toxic shock syndrome and pneumonia (lowy 1998) . it expresses several extracellular proteases with proposed roles in pathogenicity, including a serine protease v8 (sspa), cysteine proteases staphopains a and b (scpa, sspb) and a metalloprotease aureolysin (aur) (shaw et al. 2004) . staphopains a and b (family c47) are papain-like cysteine proteases that are co-expressed with their respective specific inhibitors staphostatins a and b (dubin 2003) . their role in pathogenicity has not been determined. staphopain b (sspb) is activated by the glutamyl peptidase sspa (v8 protease, family s1), which is expressed from the same operon (shaw et al. 2004 ). v8 protease modulates the surface protein profile and so influences the binding of fibronectin by staphylococcus aureus (mcgavin et al. 1997) . sortases are cysteine proteases (family c60) of grampositive bacteria that catalyse the covalent attachment of proteins to the cell wall peptidoglycan. they have been shown to contribute to the virulence of several important pathogens, including staphylococcus aureus, bacillus anthracis, listeria monocytogenes and streptococcus pneumoniae. therefore, they have been considered to be important targets for the development of novel antiinfective agents (suree et al. 2007; clancy et al. 2010) . staphylococcus aureus sortase (srta) has been at the focus of sortase inhibitor development due to the emergence of antibiotic-resistant strains and the need for novel antimicrobial strategies. several types of srta inhibitors have been investigated, including nonspecific sulfhydryl modifiers, peptide analogues of the sorting signal, compounds from plants and marine organisms, and synthetic small-molecule inhibitors. several inhibitors of srta have been described with varying strength and specificity; however, good in vitro inhibitory activity has not yet led to an effective in vivo sortase inhibitor (maresso and schneewind 2008; suree et al. 2007; clancy et al. 2010) . the cysteine protease clostripain (family c11) is a secreted protease of the anaerobic bacterium clostridium histolyticum, a causative agent of gas gangrene (clostridial myonecrosis). clostripain selectively hydrolyses arginyl bonds and constitutes an important clostridial virulence factor (jozwiak et al. 2005; manabe et al. 2010 ). in addition to oxidizing agents, thiol-blocking agents and heavy-metal ions that all inhibit clostripain, good reversible inhibitors have been described, namely, aziridine peptide esters, which are thus promising lead compounds for the development of specific clostripain inhibitors (schirmeister and peric 2000; barrett et al. 2004) . gingipains are extracellular cysteine proteases (family c25) produced by the oral pathogenic bacterium porphyromonas gingivalis, a major etiological bacterium of chronic periodontal disease. gingipains comprise two argininespecific cysteine proteases (rgpa and rgpb) and a lysinespecific cysteine protease (kgp). they constitute the major virulence factor of this periodontopathogenic bacterium as they are involved in multiple facets of its virulence and survival, including the destruction of periodontal tissues, disruption of the host immune system by inactivation of host proteinase inhibitors and deregulation of several proteinase cascades, as well as acquisition of nutrients required for bacterial growth and survival in the periodontal pocket (fitzpatrick et al. 2009; travis and potempa 2000) . due to their importance in pathogenesis, considerable efforts have been put into discovering or designing specific inhibitors of gingipains. tetracyclines, inhibitors of prokaryotic protein synthesis, have been shown to have, in addition to their antibiotic activity, inhibitory activity against cysteine proteases through binding to the proteinase outside the substrate binding site (imamura et al. 2001 ). peptidyl chloromethanes have been used as specific rgp and kgp inhibitors for their characterization (potempa et al. 1997) , and compound a71561 was shown to attenuate porphyromonas gingivalis virulence through specific kgp inhibition (curtis et al. 2002) . based on histatin cleavage specificity, small peptide analogues were designed, which specifically inhibit rgp and kgp (kyt-1 and kyt-36, respectively), which display attenuation of several virulence traits of porphyromonas gingivalis (kadowaki et al. 2004) . chlorhexidine, which has been used in oral healthcare preparations on account of its antimicrobial effects, also inhibits proteolytic activities, including those of gingipains. moreover, chlorhexidine inhibitory activity against r-gingipains was enhanced by the addition of zn(ii), which has also been used in human oral health care (cronan et al. 2006) . metalloproteases are important virulence factors of many primary and opportunistic pathogenic bacteria and cause major infectious diseases such as cholera, salmonellosis, legionnaires' disease, cystic fibrosis, botulism, tetanus and anthrax (miyoshi and shinoda 2000) . they have either direct roles in host interaction or indirect roles in processing other important virulence factors. therefore, much has been invested in the search for an effective protease inhibitor for use in treatment (jacobsen et al. 2007 ), but none has yet been developed, which would be used in clinic. metal chelators, including edta (ethylenediaminetetraacetic acid), egta (ethylene glycol-bis(2-aminoethylether)-n, n,n′,n′-tetraacetic acid) and 1,10-phenanthroline, inhibit metalloproteases in general. the ubiquitous presence of metalloproteases prevents the use of broad-spectrum inhibitors, and the search for potent and specific inhibitors of individual metalloproteases that could find clinical applications is important. the most studied bacterial metalloproteases are those of the thermolysin family (m4), including mpi protease of listeria monocytogenes, coccolysin of enterococcus faecalis, hemagglutinin/proteinase of vibrio cholerae and helicobycter pylori, pseudolysin of pseudomonas aeruginosa, aureolysin of staphylococcus aureus, legionella pneumophila protease and λ-toxin of clostridium perfringens. inhibitors of thermolysin family proteases are of bacterial origin, including those isolated from streptomyces, the small-molecule inhibitor phosphoramidon and protein inhibitor smpi (streptomyces metalloproteinase inhibitor) of family i36. another family of inhibitors targeting bacterial thermolysins is family i8 of animal origin (adekoya and sylte 2009; supuran et al. 2002; rawlings and barrett 2011) . of the bacterial metalloproteases, the light chain domains that are zinc metalloproteases (family m27) of tetanus and botulinum neurotoxins (tent and bonts) from clostridium tetani and clostridium botulinum, respectively, have also been studied extensively. various β-aminothiols have been considered as selective bont and tent inhibitors and have been further developed into strong and selective pseudotripeptide inhibitors of bont/b (blommaert et al. 2004; supuran et al. 2002) . clostridium histolyticum collagenases and their homologues from vibrio (family m9) are very effective in connective tissue degradation and hydrolyse triple helical regions of collagen under physiological conditions. they are targeted for both therapy and diagnosis of clostridial infections, and several types of compounds have been found to inhibit them. however, in addition to bacterial collagenases, they also inhibit vertebrate collagenases (supuran et al. 2002; barla et al. 2009) . no potent and selective inhibitor has yet been found for metalloproteases of family m10 from pathogenic bacteria, including serralysin from serratia, pseudomonas and erwinia, aeruginolysin from pseudomonas aeruginosa, mirabilysin from proteus mirabilis and fragilysin from bacteroides fragilis. they are, however, inhibited by protein inhibitors of bacterial origin belonging to family i38 and hydroxamate inhibitors, including batimastat (supuran et al. 2002; rawlings and barrett 2011) . another group of medicinally important bacterial proteases for which specific inhibitors have not yet been described comprises the metalloexopeptidases, which belong to several merops families (m1, m2, m14, m15, m17, m18, m19, m20, m24, m28, m29, m32, m42, m54, m55 and m61). of the metallocarboxypeptidases (belonging to families m14, m15, m20, m32) the zinc-containing d-ala-d-ala dipeptidase vanx (family m15) has been studied in view of its ability to mediate antibiotic resistance against vancomycin (crowder 2006) . similarly, the family m19 of membrane dipeptidases includes members that degrade β-lactam antibiotics. other carboxypeptidases, such as glutamate carboxypeptidases (family m20), have been studied with a view to clinical use in treating different types of cancer (holz et al. 2003) . bacterial metalloaminopeptidases, which perform essential cellular functions in protein synthesis and maintenance, have been studied as targets for novel antibiotics. a few reviews on inhibitors designed to inhibit bacterial aminopeptidases of families m17 (e.g. leucyl aminopeptidases or laps), m1 (alanyl aminopeptidase) and m24 (methionine aminopeptidases) cover the natural and designed compounds that could serve as lead compounds for inhibitors aimed at this group of proteases (mucha et al. 2010; holz et al. 2003; supuran et al. 2002; rawlings and barrett 2011) . bacterial metalloproteases that cleave immunoglobulin a (iga proteinases, families m26 and m64) constitute important colonization factors for several pathogenic bacteria (e.g. streptococcus, neisseria, haemophilus, clostridium, prevotella, capnocytophaga, bacteroides). no strong and specific inhibitor is available for these enzymes. the same is true also for another family of pathogenesis-related metalloproteases (family m23), including staphylolysin from pseudomonas aeruginosa and lysostaphin from staphylococcus simulans. in addition to their function in increasing virulence, staphylolysin and lysostaphin show bactericidal activity against staphylococcus aureus and have been studied with a view to their use in countering drug-resistant staphylococci (e.g. methicillin-and vancomycin-resistant staphylococcus aureus) (barequet et al. 2009; desbois and coote 2011) . the metalloprotease anthrax lethal factor lf (family m34) is a component of the anthrax toxin responsible for the major symptoms and death associated with bacillus anthracis infection (kim and yoon 2006 ). an increased interest in anthrax vaccination and treatment methods has been provoked by the use of bacillus anthracis spores as a bioweapon. lethal factor (lf) inhibitors would provide two-fold protection, namely, in preventing early spore protection in macrophages and, later, inhibiting lf disruption of signalling pathways through inactivation of mitogen-activated protein (map) kinase kinases. the search for an lf inhibitor to use in combination with antibiotic treatment is aimed at finding a selective and potent lf inhibitor (shoop et al. 2005; turk 2008 ), which would be non-(cyto) toxic and have good biological stability and bioavailability (johnson et al. 2009; kim et al. 2011; li et al. 2011 ). the predominant fungal diseases afflict immunocompromised patients and are caused by opportunistic pathogens candida sp. (e.g. candida albicans, candida glabrata, candida parapsilosis) and aspergillus sp. (e.g. aspergillus fumigatus, aspergillus niger, aspergillus nidulans, aspergillus calidoustus), followed by other ascomycetes of genus fusarium, basidiomycete genera malassezia, cryptococcus and trichosporon, and zygomycete genera rhizopus and mucor (boekhout et al. 2009 ). the importance of proteases in the pathogenicity of these opportunists is controversial; however aspartic, serine and metalloendopeptidases, as well as aminopeptidases, carboxypeptidases and dipeptidylpeptidases that are secreted by these species, have been proposed as the virulence factors that facilitate colonization and invasion by hydrolysis of host proteins or damage cells and molecules of the host defence system (segal 2006; yike 2011). the most studied are the secreted aspartic proteinases of candida albicans (saps) (naglik et al. 2003) . interestingly, the protease inhibitors targeted against the viral aspartic protease used in treating hiv infection also inhibited candida saps and reduced occurrence of candidiasis in these patients. secreted aspartic proteases are thus an important target for the development of new protease inhibitor based compounds for treating candidiasis (braga-silva and santos 2011; naglik et al. 2004; dash et al. 2003) . fungal proteases are also important fungal allergens, and most belong to the serine catalytic type. addition of protease inhibitor during aspergillus fumigatus and aspergillus niger protease and antigen sensitization attenuated allergic inflammation and hyper-responsiveness in an animal model (yike 2011) . secreted alkaline serine protease of aspergillus fumigatus was shown to help evade the host immune response by degrading human complement proteins, and it is therefore a good target for drug development (behnsen et al. 2010) . a network of proteolytic enzymes is very important for the survival of dermatophytes such as microsporum canis and trichophyton rubrum, the specialized pathogenic fungi that infect stratum corneum, nails or hair of healthy individuals. it includes the metalloendopeptidases, fungalysins (family m36), serine endopeptidases, subtilisins (family s8a) and several exopeptidases (dipeptidyl peptidases (family s9), aminopeptidases (family m28) and carboxypeptidases (family s10)) (monod 2008) . although proteases are only one of the several groups of virulence factors of pathogenic fungi, they aid in the invasion of tissues and evasion of immune responses. therefore, specific protease inhibitors aimed at them would constitute a valuable addition to the presently used antifungal drugs that target fungal cell wall and cell membrane integrity or dna replication-and to which many pathogenic strains have acquired resistance (marie and white 2009) . the classspecific (broad-spectrum) aspartic protease inhibitor pepstatin has been shown to inhibit adhesion of candida albicans and prevent invasion or mucosal tissue damage by inhibiting the saps (naglik et al. 2004) . recently, it has been shown that ergosterol production transcriptional regulator (sre1), which is activated by a metalloprotease stp1, is essential for the survival of cryptococcus neoformans in the presence of antifungal drugs that inhibit sterol biosynthesis. therefore, regulators of the ergosterol pathway, including the metalloprotease stp1, constitute promising targets for novel antifungal therapeutics to be used in combination with a sterol synthesis inhibitor for treating cryptococcosis in immunocompromised individuals (bien et al. 2009 ). protozoan parasitic diseases, including malaria, leishmaniasis and african and american trypanosomiasis, are some of the most important infectious diseases in the world, with high mortality and morbidity rates in developing countries. reasons for their persistence, despite prolonged use of antiparasitic drugs, include their toxic side effects and the increasing emergence of drug resistance. therefore, research over the past 15 years has been focused on identifying new targets for antiparasite treatment and on developing substances suitable for human therapy. parasitic proteases constitute one of the very important druggable targets since they are key virulence factors due to their essential roles in cell metabolism and interaction with the host (zucca and savoia 2011; renslo and mckerrow 2006; mckerrow et al. 2008) . aspartic proteases plasmepsins (family a1), which are important for haemoglobin catabolism in parasites causing malaria (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae), have been targeted for the design of novel antimalarial drugs, i.e. selective protease inhibitors. although plasmepsin inhibitors (including the broad-spectrum pepstatin) have been shown to have antimalarial effects, the main challenge still remains to design an inhibitor that would be active against different plasmepsins i, ii and iv, and the histoaspartic protease hap involved in haemoglobin degradation, but also inactive against the homologous human aspartic proteases (cathepsins d and e) (zucca and savoia 2011; rosenthal 2003; ersmark et al. 2006; gil et al. 2011 ). in addition to plasmepsins, the cysteine proteases falcipains (family c1), metalloprotease falcilysin (family m16) and aminopeptidases have been targeted for development of new antimalarial protease inhibitors. a synergistic effect has been observed for their combined use (e.g. a cocktail of aspartic and cysteine protease inhibitor) (mckerrow et al. 2008; rosenthal 2003; zucca and savoia 2011; trenholme et al. 2010) . cysteine proteases similar to falcipain (family c1) have been associated with the pathogenesis of trypanosomiasescruzipain of trypanosoma cruzi (chagas disease) and brucipain or rhodesain of trypanosoma brucei (sleeping sickness). cruzipain (or cruzain) has been extensively studied and is considered to be a promising target for chemotherapy since it is critical for parasite viability in all stages of infection, especially for nutrition acquisition, tissue invasion and host immune response evasion (duschak and couto 2009 ). an irreversible inhibitor, k777, was effective in pre-clinical models of chagas disease; however, a safer treatment would be achieved by utilizing a reversible and highly specific cruzipain inhibitor (beaulieu et al. 2010; mckerrow et al. 2009; brak et al. 2010 ). cysteine proteases have been implicated in the pathogenesis of leishmania species and are major virulence factors as they substantially modify the immune response. a surface metalloprotease gp63 or leishmanolysin (family m8) has been shown to be essential for establishing and maintaining the infection and would make a promising target for development of a selective protease inhibitor for antiparasitic chemotherapy (olivier and hassani 2010; yao 2010) . in addition to the computational design, development and optimization of a suitable protease inhibitor, based on the 3d structure of the target protease, the search for novel types of inhibitors from natural sources (pereira et al. 2011) , such as fungi and microbes, is important for identifying new lead compounds. serine proteases are also a neglected group of potentially targetable protozoan proteases involved in their pathogenicity, although several subtilisins (family s8) have been described from plasmodium falciparum and toxoplasma gondii, and two oligopeptidases (family s9) from trypanosoma cruzi (mckerrow et al. 2008) . in addition to protozoan parasites, helminths, which include parasitic roundworms (nematodes) and flatworms (trematodes and cestodes), also cause important parasitic infections. cysteine cathepsins (family c1), which are important for many aspects of the helminth-host relationship, are a potential target for developing antihelminthic drugs. cysteine protease inhibitors have been shown to impair fecundity of the liver fluke (fasciola hepatica) and blood fluke (schistosoma mansoni) in animal models. however, the similarity of host cathepsins, together with their importance, calls for the design of a very specific and selective protease inhibitor. alternatively, drug design could exploit bioavailability and pharmaco-kinetic and -dynamic properties to target parasitic proteases preferentially (robinson et al. 2008 ). in addition to proteases, parasite-derived protease inhibitors play important roles in manipulating the host immune system and establishing a niche for the successful feeding and reproduction of helminth parasites. therefore, protease inhibitors are also important targets for immunological control of helminth parasitic infections and make proteases that are insensitive to parasite-derived protease inhibitors valuable candidates for new types of antihelminthic therapy (knox 2007; stepek et al. 2006) . the ability of tumour cells to invade extracellular barriers and to metastasize to distant sites is associated with the activity of proteases (kos and lah 1998) . the major molecular mechanism, which involves the active role of various intra-and extracellular proteases, is the dissolution and remodelling of connective tissue and the basement membrane. it includes matrix metalloproteases (mmp), serine proteinases such as urokinase, tissue types of plasminogen activator (upa, tpa) and plasmin, aspartic proteinase cathepsin d and cysteine proteinases cathepsins b, h, s and l (schmitt et al. 1992 ). in addition to extracellular matrix remodelling, proteases regulate several other processes, leading to the progression of malignant disease, such as cell adhesion, migration, differentiation, proliferation and signalling of tumour cells. it is well accepted that tumourassociated proteolytic activity escapes the control of endogenous protease inhibitors and that treatment of patients with exogenous protease inhibitors may suppress the progression of the disease and improve the outcome of cancer patients. however, the treatment should specifically target the tumour-associated proteases that cause harmful actions and not those involved in the numerous physiological processes in cells and tissues. several protease inhibitors failed in clinical trials due to lack of specificity, resulting in severe side effects and/or lack of clinical benefit in treated patients (turk 2006; coussens et al. 2002) . new approaches to developing protease inhibitors applicable in therapeutic interventions include structure-based medicinal chemistry and development of molecular systems to deliver inhibitors to the site of action. among the small-molecule inhibitors of bacterial and fungal origin, peptidyl aldehydes such as leupeptin and antipain, hexapeptide pepstatin and epoxysuccinyl peptide e-64 and their analogues have been studied as anticancer agents. the thiol-protease specific inhibitor, e-64, originally isolated from aspergillus japonicus (hanada et al. 1978) , has been studied extensively as a potential antitumour agent in cell culture and animal models. derivatives of e-64, displaying selectivity between different cysteine proteases (frlan and gobec 2006) , represent the next step towards their application in treating cancer and other diseases. they were designed on the basis of the x-ray crystal structures of individual cathepsins, and the most studied were cathepsin b specific inhibitors ca-074 and ca-030, cathepsin l specific inhibitors clik-148 and clik-195, and cathepsin x specific inhibitor ams-36. cathepsin s specific inhibitor clik-060 was designed on the basis of the structure of leupeptin and antipain (katunuma 2011) . antitumour activity was exhibited particularly by ca-074, a specific inhibitor of the cysteine protease cathepsin b (johansson et al. 2000) , which appears to be crucial for tumour cell invasion (lah et al. 2006) . in animal models, it was shown that ca-074 reduces tumour growth, invasion and angiogenesis of many cancer types, including pancreatic cancer, melanoma and breast tumours, all tested on animal models. the cell permeability of epoxysuccinyl inhibitors was improved by esterification. the esters are less active than free acids; however, in cells, they are rapidly hydrolysed to their active form. better cell permeability was demonstrated for ethyl ester e-64d and the methyl ester of ca-074, which are also highly soluble and effective for prolonged periods (frlan and gobec 2006) . metalloproteases are an important group of proteases that have been considered extensively as targets for cancer therapy due to the many roles they play in carcinogenesis and tumour invasion, growth and dispersion. however, the diversity of endogenous metalloproteases and their numerous and versatile physiological roles have prevented the use of broad-spectrum metalloprotease inhibitors. much effort is invested in determining which metalloproteases to target and in designing highly selective and potent protease inhibitors based on the structural characteristics of individual target metalloproteases (coussens et al. 2002; bialas and kafarski 2009; dorman et al. 2010; overall and kleifeld 2006) . several proteases of the serine catalytic type have also been targeted for the design of specific protease inhibitors for use in cancer treatment, including the urokinase plasminogen activator and matriptase (abbenante and fairlie 2005; bialas and kafarski 2009; ulisse et al. 2009 ). the broad-range microbial inhibitors of serine, cysteine and threonine proteases, leupeptin and antipain, were also shown to inhibit malignant transformation (vaccari et al. 1999) or tumourigenesis (hozumi et al. 1972) . threonine catalytic type proteolysis is present in proteasomes, and several chemical classes of natural and synthetic proteasome inhibitors have been considered as anticancer agents because of their preferential antiproliferative and proapoptotic activity on cancer cells (kisselev and goldberg 2001; abbenante and fairlie 2005; bialas and kafarski 2009; cecarini et al. 2011; chen et al. 2011) . the aspartic protease inhibitor pepstatin has also been frequently tested as an antitumour agent since cathepsin d was identified as an important tumour promoting factor (greenbaum and sutherland 1983; benes et al. 2008 ). irregular function of proteases is associated with a variety of other diseases, representing targets for therapeutic application of all catalytic types of protease inhibitors. smallmolecule inhibitors of bacterial and fungal origin, described already in the previous section, particularly epoxysuccinyl inhibitors, have been reported as potential protective agents in autoimmune, neurodegenerative, antiinflammatory and cardiovascular diseases; osteoporosis; muscular dystrophy; diabetes and others. ca-074 and clik-148 were demonstrated to cause a switch between th1 and th2 t cell response due to the different roles of cysteine cathepsins b and l in antigen processing and presentation (katunuma 1997) . cathepsin s inhibitor clik-060 has been shown to suppress sj gren syndrome, an autoimmune disease associated with the processing of α-foldin by cathepsin s (saegusa et al. 2002) . the cathepsin x inhibitor ams-36 reduces the activation of integrin receptor lfa-1 (lymphocyte functionassociated antigen 1), a molecule involved in t cell adhesion, proliferation and migration (jevnikar et al. 2008) . lfa-1 overexpression and activation by cathepsin x is typical of autoimmune diseases, particularly psoriasis. the same inhibitor significantly enhanced the proliferation of neuronal cells and neuritogenesis, preventing the processing of neurotrophic factor γ-enolase by cathepsin x (obermajer et al. 2009 ). epoxysuccinyl inhibitors have been tested in several animal models of neurodegenerative diseases. e-64 was shown to restore normal synaptic function in the app/ps1 mouse model of alzheimer's disease with overexpressed amyloid precursor protein (app) and presenilin 1 (ps1) (trinchese et al. 2008 ). e-64d and ca-074me also reduced the accumulation of neurotoxic beta-amyloid peptides in brains, presumably inhibiting cathepsin b involved in processing the amyloid precursor protein (hook et al. 2007 ). aspartic proteases β-secretase (also known as memapsin-2 or bace1 and belonging to merops family a1) and γ-secretase (composed of two presenilins belonging to merops family a22) are important therapeutic targets for treating alzheimer's disease, and several compounds designed to reduce their activity are in clinical trials (de strooper et al. 2010) . another important target of aspartic proteases is renin (family a1), which is part of the complex renin-angiotensin-aldosterone system that regulates blood pressure and electrolyte balance. several inhibitors have been designed based on the renin structure and activity, and one of them, aliskiren, is the first non-peptide, orally administered, direct renin inhibitor available on the market for management of hypertension (nguyen et al. 2008; barrios and escobar 2010) . the very first protease inhibitor used in humans as a therapeutic agent, namely, an inhibitor of metalloprotease angiotensin-converting enzyme (ace), was also hypertension related. it is an important regulator of the reninangiotensin system, and ace inhibitors are used for treating hypertension, but are also implicated in other cardiovascular and renal diseases (abbenante and fairlie 2005; ondetti et al. 1977) . the inhibitors of collagenolytic enzymes, such as cathepsins l and k, prevent bone remodelling and can be useful in treatment of osteoporosis. several synthetic cathepsin k inhibitors are in different stages of clinical trials. in addition to osteoporosis, they are also considered for application in arthritis, atherosclerosis, obesity and cancer (bromme and lecaille 2009) . administration of cathepsin l inhibitor clik-148 significantly reduced invasion and metastasis formation in bones (katunuma 2011) . e64d, a methyl ester of e64, was tested for treating muscular dystrophy; however, its further development was stopped in phase iii due to insufficient effectiveness and hepatic injury in rats (fukushima et al. 1990 ). antipain, leupeptin and pepstatin have also been tested for treatment of muscular dystrophy; however, persuasive benefit was not demonstrated in animal models. specific inhibitors designed to target the serine aminopeptidase dipeptidyl peptidase iv are in clinical trials for management of type 2 diabetes (abbenante and fairlie 2005; tahrani et al. 2011) . endogenous plant protease inhibitors constitute one of the plant defence strategies against pathogenic, parasitic and herbivorous organisms. they target the important proteolytic virulence factors of phytopathogenic bacteria, fungi, parasites and viruses, preventing their roles in nutrient acquisition and evasion of host defence. furthermore, they target digestive proteases of herbivorous pests (e.g. insects, mites, slugs), preventing the utilization of food-derived organic nitrogen for their growth and development (ryan 1990; haq et al. 2004) . since pests and pathogens depend on utilization of these proteases, there is a strong selection pressure operating to develop resistance to plant endogenous defensive protease inhibitors (haq et al. 2004; jongsma and beekwilder 2008) . therefore, the search for novel protease inhibitors with potential protective function is very important for the development of environmentally friendly pest and pathogen management strategies. in addition to investigations aimed at augmenting crop endogenous resistance by conventional breeding, there are several protease inhibitors of plant origin that have been used in the preparation of genetically modified crop plants with superior ability for biotic stress resistance (ferry and gatehouse 2010) . the use of protease inhibitors for insect pest management has gained most attention. insect pests cause major economic losses annually, of which lepidopteran (butterflies' and moths') larvae are considered the most destructive. agricultural pests causing significant economic impact also belong to orders coleoptera (beetles), diptera (true flies), hemiptera (e.g. aphids), orthoptera (e.g. locust) and thysanoptera (thrips). they cause either direct damage to crops by feeding or indirect damage by transmitting viral diseases or secondary microbial infections. the most notable for their destructive capacity are the migratory locust (locusta migratoria), several beetles, including colorado potato beetle (leptinotarsa decemlineata), boll weevil (anthonomus grandis), japanese beetle (popillia japonica), the western corn rootworm (diabrotica virgifera virgifera) and many species of aphids belonging to all families of the superfamily aphidoidea. different catalytic types of proteases provide the predominant proteolytic activity in different groups of insect pests. while serine proteases are predominant in digestive proteolysis in most insect species (e.g. lepidoptera, diptera), cysteine proteases predominate in hemiptera, coleoptera and thysanoptera. in addition, aspartic and metalloproteases complement protein digestion to different degrees in most insect orders (terra and ferreira 1994) . therefore, protease inhibitors targeting different groups of proteases have shown variable antinutritional effects when fed to different insect pests. the catalytic, classspecific, small-molecule protease inhibitors of microbial origin have often been used for proof-of-principle feeding experiments, but protein protease inhibitors were then employed to generate insect-resistant transgenic plants. these were predominantly of plant origin, with a few exceptions of animal-derived protease inhibitor genes (haq et al. 2004; dunaevsky et al. 2005; malone et al. 2008) . other important proteins that have been used for constructing pest-resistant transgenic crops are insecticidal proteins derived from bacillus thuringiensis (bt), and genetically modified maize and cotton varieties that express bt toxins have become an important component in agriculture worldwide and have reduced the use of pesticides and lowered production costs (ferry and gatehouse 2010; kumar et al. 2008) . the involvement of endogenous protease inhibitors in natural plant resistance against herbivores is probably the basis of the adaptation of lepidopteran and coleopteran species to ingestion of protease inhibitors that has been observed for several species (jongsma and beekwilder 2008; wu et al. 1997; bonade-bottino et al. 1999; lara et al. 2000) . they circumvent the antinutritional effect of the ingested protease inhibitors either by overexpression of native gut proteases or by production of insensitive proteases; some of which can degrade the ingested protease inhibitors (ferry and gatehouse 2010; jongsma and beekwilder 2008) . therefore, the pyramiding or stacking of different families of inhibitors to increase the spectrum of inhibitory activity has been shown to have synergistic effects, as well as combining protease inhibitor genes with genes of other insecticidal proteins, namely, lectins, bt or other bacterial toxins and α-amylase inhibitors (ferry and gatehouse 2010; schluter et al. 2010; christou et al. 2006; malone et al. 2008) . furthermore, the use of protease inhibitors of microbial and fungal origin could offer superior characteristics, such as stability, resistance to proteolytic degradation and diverse inhibitory patterns, for a more potent antinutritional or insecticidal effect. only a few examples of utilization of microbial small-molecule inhibitors as antinutritional agents are available, e.g. aminopeptidase inhibitors of actinomycetes amastatin and bestatin against the red flour beetle (tribolium castaneum) (oppert et al. 2011) , aspartic protease inhibitor pepstatin a from actinomycetes against the cowpea bruchid (callosobruchus maculatus) (amirhusin et al. 2007) , the serine and cysteine protease inhibitor leupeptin from actinomycetes against western corn rootworm (diabrotica virgifera) (kim and mullin 2003) and cysteine protease inhibitor e-64 from aspergillus japonicus against colorado potato beetle (leptinotarsa decemlineata) (bolter and latoszekgreen 1997) . the use of protein protease inhibitors is advantageous since transgenic plants expressing a pest resistance gene in a controlled manner represent a stable and cheap propagation source that would lower the amount of pesticides needed, making plant protection environment friendly. to our knowledge, the only examples of a protein protease inhibitor of microbial or fungal origin as an effective antinutritional agent are the cysteine protease inhibitors macrocypins (family i85) from the edible parasol mushroom (macrolepiota procera), which have been shown to be detrimental to the growth and development of colorado potato beetle larvae (istinič et al. 2011) . the high capacity for development of resistance to insecticidal proteins in major insect pests drives the search for effective protease inhibitors. novel protein protease inhibitors aimed at serine proteases would be useful for management of major agricultural pests such as beet armyworm (spodoptera exigua), cotton bollworm (helicoverpa armigera and helicoverpa zea) and tobacco hornworm (manduca sexta). a combination of serine and cysteine protease inhibitors would be useful against, e.g. boll weevil (anthonomus grandis), cowpea weevil (callosobruchus maculatus) and red flour beetle (tribolium castaneum), and cysteine protease inhibitors would, for example, be useful against the colorado potato beetle (leptinotarsa decemlineata), western corn rootworm (diabrotica virgifera), banana weevil (cosmopolites sordidus) and pea aphid (acyrtosiphon pisum). microorganisms that are generally recognized as safe and edible mushrooms offer a valuable source of such novel protease inhibitors that would also be acceptable for use in crops for human consumption. in addition to insect pests, other herbivores causing significant crop losses worldwide include mites and slugs. in both cases, cysteine proteases constitute the predominant digestive proteolytic activity. plant cystatins have been shown to be detrimental to mite development and reproductive performance in feeding trials on one of the major mite pests on agricultural crops, the two-spotted spider mite tetranychus urticae (acari: tetranychidae) (carrillo et al. 2011) . similarly, the growth of juvenile slugs deroceras reticulatum, the important agricultural and horticultural pest, was significantly reduced when fed with leaf tissue overexpressing a plant cysteine protease inhibitor (walker et al. 1999) . therefore, protease inhibitors of microbial and fungal origin have great potential for protecting plants from important mite pests and suppressing the growth rates of slug populations, in addition to their antinutritional characteristics for different insect pests. another advantage of the application of protease inhibitors in crop protection is that in addition to protection against herbivorous pests, they offer cumulative protection against nematodes and bacterial, fungal and viral pathogens (haq et al. 2004 ). the use of protease inhibitors is one of many strategies to protect plants from parasitic nematodes. their targets are intestinal proteases, mostly of the cysteine catalytic class. therefore, heterologously expressed plant cysteine protease inhibitorsphytocystatins (family i25)-have been effective to different degrees against beet-cyst nematode (heterodera schachtii), root-knot nematode (meloidogyne incognita), potato cyst nematode (globodera pallida) and burrowing nematode (radopholus similis) (mccarter 2009; haq et al. 2004) . a plant-derived serine protease inhibitor offered satisfactory nematode resistance in transgenic wheat against the cereal cyst nematode (heterodera avenae) (vishnudasan et al. 2005; mccarter 2009 ). the antifungal effect of serine protease inhibitors has been determined with plant-derived protease inhibitors that target secreted proteases (mainly families s1 and s8) of phytopathogenic fungi involved in plant cell wall penetration by hyphae (wong et al. 2010 ). in addition, aspartic and cysteine proteases that are also fairly widespread (valueva and mosolov 2004) represent potential targets for protective protease inhibitors. similarly, phytopathogenic bacteria secrete proteases involved in pathogenesis, mainly aiding plant cell wall degradation. together with pectinases, cellulases and hemicellulases, they contribute to massive degeneration of plant tissue, for example, in wilt (e.g. ralstonia solanacearum) and soft-rot diseases (e.g. erwinia chrysanthemi, erwinia carotovora) (kunkel and chen 2006) . cysteine proteases belonging to families c48, c55, c58, c70 and c72, which have been implicated in the pathogenicity of many phytopathogens belonging to genera pseudomonas, xantomonas, ralstonia, erwinia and pantoea as effectors of the type iii secretion system, constitute potential targets for protective protease inhibitors (kunkel and chen 2006; mosolov and valueva 2006) . the importance of polyprotein processing in replication of viruses, especially those of families potyviridae and comoviridae, indicates that protease inhibitors could mediate resistance to plant viruses by inhibiting target viral proteases. indeed, a rice cystatin (oryzacystatin) expressed in tobacco plants conferred resistance to potato virus y (pvy) and to tobacco etch virus (tev) which correlated to increased inhibition of the target papain-like protease sudarshana et al. 2007 ). in addition to their protective role against biotic stress, endogenous plant protease inhibitors have been implicated in the control of proteolytic systems in plants under abiotic stresses with a dehydration component such as drought, increased salt concentration and freezing (vaseva et al. 2012) . it has been shown that the changes in metabolism triggered by water deficit involve active involvement of regulated proteolysis that assists the protective proteins (dehydrins and chaperones) in the cellular response to the increased levels of denatured, aggregated or oxidatively damaged proteins that accumulate during dehydration stress (brzin and kidrič 1995; benchabane et al. 2010; bray 1993; hoekstra et al. 2001; feller 2004) . overexpression of endogenous protease inhibitors increased resistance to drought stress, as shown for overexpression of the cysteine protease inhibitors cystatins atcys1 and atcys2 in the model plant arabidopsis thaliana ) and for overexpression of a serine protease inhibitor ocpi1 (oryza sativa chymotrypsin inhibitor 1) in rice, with the latter showing improved drought resistance in terms of yield loss in the field (huang et al. 2007) . although protease inhibitors have great potential in protecting crops under abiotic stress conditions, detailed knowledge of the specific roles of different proteases in response to abiotic stress is needed before exogenous protease inhibitors can be used to manipulate and improve drought resistance in plants (vaseva et al. 2012) . small-molecule protease inhibitors are routinely used as buffer additives when preparing protein extracts, in order to prevent proteolytic degradation during protein purification procedures. broad-spectrum inhibitors that cover all the different catalytic classes are generally used, including pepstatin a for aspartic proteases, e-64 for cysteine proteases, chymostatin for serine proteases, antipain for cysteine and serine proteases, and leupeptin for cysteine, serine and threonine proteases; all of which were originally isolated from actinomycetes. for inhibition of serine proteases, synthetic protease inhibitors such as aebsf (pefabloc; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride), the physiologically more stable derivative of pmsf (phenylmethanesulfonyl fluoride), are used. chelating agents such as edta and 1,10-phenanthroline are generally used to inhibit metalloproteases and, for certain applications, inhibitors of actinomycete origin-phosphoramidon (m4 and m13 metallopeptidases inhibited) or bestatin (aminopeptidases inhibited). unwanted proteolytic degradation can cause severe reduction in yield of heterologously expressed proteins in various expression systems. therefore, natural or engineered protease-deficient strains are often used as hosts for bacterial (e.g. escherichia coli bl21), yeast (e.g. pichia pastoris smd 1163, 1165 or 1168) and filamentous fungal (e.g. aspergillus niger prt pep) expression systems. however, these strains may not exhibit optimal bioprocessing characteristics due to lower fitness traits. alternative strategies are therefore used for preventing proteolytic degradation of recombinant proteins, including modification of the recombinant protein sequence to remove protease cleavage sites without affecting protein function, expression with a stabilizing fusion partner, optimization of cultivation conditions (ph, temperature, medium composition, bioprocess strategy) and use of protease inhibitors. for secreted recombinant proteins, small-molecule inhibitors can be added to the culture medium to inhibit the predominant secreted proteolytic activity of the host organism that is often of the serine and aspartic catalytic type. another strategy is coexpression of an appropriate protein protease inhibitor with the recombinant protein, which may, however, influence the yield or complicate the downstream purification procedures (potvin et al. 2010; sharma et al. 2009; rozkov and enfors 2004) . in addition to protection against proteolytic degradation of recombinant protein during the expression process, protease inhibitors as fusion partners have other advantages. one example is the use of the bacterial periplasmic serine protease inhibitor ecotin (family i11) as a vehicle for secretion to the periplasmic space of escherichia coli (paal et al. 2009 ). another example is the serine protease inhibitor from oyster mushroom, the pleurotus ostreatus proteinase a inhibitor 1 (poia1) (family i9) that serves as an intramolecular chaperone enabling proper refolding of the fused subtilisin protease from inclusion bodies expressed in a heterologous bacterial expression system (kojima et al. 2005) . the strategy of using co-expression of protein protease inhibitors for reducing proteolytic degradation of heterologously expressed proteins has been successfully implemented in plant expression systems. preparation of a protease-deficient host is, in this case, not applicable because of the essential roles of proteases in growth and development. plant expression systems offer many advantages over bacterial and yeast expression systems (e.g. posttranslational modification capability) and over animal cell lines (e.g. lower cost and contamination risks) for largescale recombinant protein production, but have not yet been commercialized due to low levels of protein expression and of heterologous protein accumulation. the latter can be influenced by targeting the expression to specific organelles (e.g. endoplasmic reticulum) or tissues (e.g. seeds and tubers) or by co-expression with stabilizing fusion partners or protease inhibitors. co-expression of protective protease inhibitors does not have any adverse effects on plant growth and development, as described previously for examples of protease inhibitor expressing, insect-resistant transgenic plants. furthermore, the heterologously expressed protease inhibitors offer the added advantage of protection against proteolysis also ex planta, especially in the early steps of purification of crude protein extracts, thus minimizing the need for addition of protease inhibitors to the extraction buffers (rivard et al. 2006; desai et al. 2010; doran 2006; benchabane et al. 2008) . so far, only protease inhibitors of plant and animal origin have been used as co-expression partners; use of microbial or fungal protease inhibitors could offer superior protective characteristics. however, the properties of the protein of interest and of the selected host plant species influence the selection of an appropriate protective protease inhibitor, and the choice still has to be made on a case-by-case basis. another important biotechnological application of protease inhibitors is in protein purification, where they can be used as ligands in affinity chromatography. affinity chromatography is a simple one-step purification method of a molecule from a complex mixture based on specific and high affinity binding to a ligand immobilized on a solid support. reversible protease inhibitors of microbial origin are excellent ligands for purification of their cognate proteases by affinity chromatography. depending on the target protease, inhibitors with broad range or very specific inhibitory spectrum can be selected for a ligand. attention must be paid to the strength of the inhibitor as purification is not possible when the binding is too weak (no binding) or too strong (ineffective elution) (ge 2007) . the advantage of using microbial and fungal protease inhibitors is that many of them display unique inhibitory profiles and resistance to proteolytic cleavage, as well as high thermal and broad ph range stability, with the latter being very convenient since harsh conditions may be used for immobilization to the matrix as well as for the several cycles of elution steps, usually involving extreme change in ph and/or ionic strength. the broad-range inhibitor of pepsin-like aspartic proteases, pepstatin a, has been used for purification of aspartic proteases from several different sources, including higher fungi (sabotič et al. 2009a) , plants (payie et al. 2000) and insect recombinant enzyme expressed in a bacterial expression system (volkov et al. 2004) . several different types of inhibitor have been used for purification of serine proteases, including the synthetic inhibitor benzamidine and plant-and animal-derived protease inhibitors of 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discovery of dengue protease inhibitors bacterial proteinases as targets for the development of second-generation antibiotics aminopeptidases of malaria parasites: new targets for chemotherapy inhibition of calpains improves memory and synaptic transmission in a mouse model of alzheimer disease purification and characterization of streptomyces griseus metalloendopeptidases i and ii targeting proteases: successes, failures and future prospects discovery and development of anthrax lethal factor metalloproteinase inhibitors the urokinase plasminogen activator system: a target for anti-cancer therapy pepstatin, a new pepsin inhibitor produced by actinomycetes effects of the protease inhibitor antipain on cell malignant transformation thrombin-activatable fibrinolysis inhibitor is degraded by salmonella enterica and yersinia pestis role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms plant proteases: from phenotypes to molecular mechanisms the cladosporium fulvum virulence protein avr2 inhibits host proteases required for basal defense the response of plants to drought stress-the role of dehydrins, chaperones, proteases and protease inhibitors in maintaining cellular protein function molecular cloning, over expression, and activity studies of a peptidic hiv-1 protease inhibitor: designed synthetic gene to functional recombinant peptide enzymatic characterization of the streptococcal endopeptidase, ides, reveals that it is a cysteine protease with strict specificity for igg cleavage due to exosite binding assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (pin2) gene new aspartic proteinase of ulysses retrotransposon from drosophila virilis transgenic arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug deroceras reticulatum (muller) picornaviral 3c protease inhibitors and the dual 3c protease/coronaviral 3c-like protease inhibitors regulation of bacterial protease activity proteins with antifungal properties and other medicinal applications from plants and mushrooms adaptation of helicoverpa armigera (lepidoptera: noctuidae) to a proteinase inhibitor expressed in transgenic tobacco novel inhibitor for prolyl tripeptidyl aminopeptidase from porphyromonas gingivalis and details of substrate-recognition mechanism major surface protease of trypanosomatids: one size fits all? fungal proteases and their pathophysiological effects proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins two cysteine proteinase inhibitors from arabidopsis thaliana, atcysa and atcysb, increasing the salt, drought, oxidation and cold tolerance current developments in the therapy of protozoan infections acknowledgements we are grateful to dr. jože brzin for numerous discussions and suggestions for the improvement of the review and to professor roger h. pain for critical reading of the manuscript and language editing. we would like to thank dr. miha renko for the assistance with fig. 1. key: cord-012066-8anhcyia authors: stams, alfons j. m.; huisman, jacco; garcia encina, pedro a.; muyzer, gerard title: citric acid wastewater as electron donor for biological sulfate reduction date: 2009-07-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-009-1995-7 sha: doc_id: 12066 cord_uid: 8anhcyia citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. the pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. citrate was not a direct electron donor for the sulfate-reducing bacteria. instead, citrate was fermented to mainly acetate and formate. these fermentation products served as electron donors for the sulfate-reducing bacteria. sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. two citrate-fermenting bacteria were isolated. strain r210 was closest related to trichococcus pasteurii (99.5% ribosomal rna (rrna) gene sequence similarity). the closest relative of strain s101 was veillonella montepellierensis with an rrna gene sequence similarity of 96.7%. both strains had a complementary substrate range. the biological sulfur cycle plays an important role in nature. in addition, the biological sulfur cycle can be applied in biotechnology to remove and recover sulfur from wastewater and gas (buisman et al. 1989; janssen et al. 1995a janssen et al. , 2001 lens et al. 1998; muyzer and stams 2008) . chemolithotrophic sulfide-oxidizing bacteria oxidize sulfide to sulfate, but under oxygen-limiting conditions, mainly elemental sulfur is formed (janssen et al. 1995a ). this property of sulfide-oxidizing bacteria is applied to convert hydrogen sulfide in biogas or natural gas to elemental sulfur (janssen et al. 2001) . presently, about 100 of such full-scale installations are in operation worldwide. biological processes can also be applied for the removal of sulfate from wastewater (buisman et al. 1989; johnson 2000; lens et al. 1998 ). the sulfate-containing stream is led into an anaerobic bioreactor, in which, by the activity of dissimilatory sulfatereducing bacteria, hydrogen sulfide is formed. in a second micro-aerobic bioreactor, sulfide-oxidizing bacteria then oxidize sulfide to elemental sulfur. this biological sulfate removal process is an attractive and economical feasible alternative for the well-known lime process for flue gas desulfurization, in which gypsum is formed (hulshoff pol et al. 2001; janssen et al. 2001) . presently, a full-scale biological sulfate removal installation is in operation with a capacity to produce 12 t of sulfur per day (www.paques.nl). the biological process is operated at low temperature, and a citrate-containing wastewater stream is used as electron donor for biological sulfate reduction. dissimilatory sulfate-reducing bacteria are able to use a wide variety of organic compounds as electron donor for sulfate reduction (hansen 1994; muyzer and stams 2008; widdel and hansen 1992) . hydrogen, formate, lactate, malate, and ethanol are well known substrates for desulfovibrio, desulfomicrobium, and desulfotomaculum species. in general, these bacteria oxidize organic compounds incompletely to acetate. short chain and long chain fatty acids are substrates for different genera of sulfate-reducing bacteria like desulfobulbus, desulfococcus, desulfosarcina, and desulfonema. acetate is only a good electron donor for some specialized bacteria like desulfobacter postgatei and desulfobacca acetoxidans (dar et al. 2007; oude elferink et al. 1994 , 1999 . hydrogen-rich gas is being used as electron donors for biological sulfate reduction at low temperature at full scale (van houten et al. 2006; weijma et al. 2002) , while at moderately thermophilic conditions (65°c), methanol was found to be an excellent electron donor for biological sulfate reduction as well weijma et al. 2000) . in a recent study, methanogenesis and sulfate reduction with citrate was studied (gámez et al. 2008 ), but citrate is not a known common substrate for sulfate-reducing bacteria. in fact, citrate is rarely tested as growth substrate for newly isolated species. desulfovibrio oxamicus is able to grow with citrate (lópez-cortés et al. 2006) , while desulfomicrobium apsheronum was tested but was not able to grow with citrate (rozanova et al. 1988) . the aim of the present study was to elucidate the pathway of citrate conversion coupled to sulfate reduction in the above-mentioned fullscale bioreactor and to identify the microorganisms involved. we mainly focused our research on the conversion of citrate and the microorganisms involved. the sulfatereducing community of the starting sludge had been analyzed previously (dar et al. 2007) . this sludge contained different types of sulfate-reducing bacteria, including bacteria from the genera desulfovibrio/desulfomicrobium, desulfobulbus, and desulfobacca. the samples originate from a biological sulfate reduction installation at yixing (people's republic of china). the sulfate-reducing bioreactors were started up with sludge from a sulfate-reducing bioreactor from a chemical plant located at emmen, the netherlands. the installation is operated at low temperature. the wastewater to be treated is mixed with a citrate-containing waste stream and fed to three anaerobic bioreactors, in which sulfate is reduced to sulfide. the granular sludge bed reactors are equipped with internal settlers for biomass retention. they are operated at a hydraulic retention time of approximately 6 h and an influent flow rate of around 800 m 3 /h, depending on the availability of the wastewater. the total reactor volume was approximately 5,000 m 3 . there is no active sludge management (e.g., sludge harvesting) as the slow growth rate of anaerobic bacteria results in a small biomass production. the biomass entered with the feed stream and was washed-out with the effluent. consequently, a reliable sludge age cannot be determined. the effluent sulfide concentration of the anaerobic bioreactors is maintained at 150 mg/l or higher. methanogenesis did not occur. in the overall process, the dissolved sulfide is converted into elemental sulfur in an aerobic bioreactor with air as oxidation agent. the elemental sulfur is removed from the liquid by gravity settling. these two steps are not of relevance for this study. two sludges from the full-scale reactors were used in this study; sludge s was taken near the influent point of the reactor, while sludge r was from the central part of the reactor. the biomass in the reactor was not homogeneous in appearance. sludge r had a more compact structure than sludge s. the dry weight content of sludge r and sludge s was about 3.9% and 0.8%, respectively, and the volatile suspended solids (vss, a measure of organic carbon) content was about 3.0% and 0.2%, respectively. the differences in dry weight and vss content of the sludges corresponded rather well with the difference in cell numbers determined by direct counting by microscopy. the cell numbers were about 3.10 11 and 5.10 10 cells per milliliter for sludge r and s, respectively. a bicarbonate-buffered mineral medium supplemented with 0.05 g/l yeast extract was prepared as described previously (stams et al. 1993 ). this medium was used for the activity tests with the sludges and for the enrichment and description of the bacteria. routine cultivation of the strains was carried out in 117-ml serum bottles with 50 ml medium or in 28-ml tubes with 10 ml medium, and a n 2 /co 2 (80:20) gas phase at a pressure of 170 kpa. organic substrates were supplied at a concentration of 10 or 20 mm and where indicated sulfate at a concentration of 20 mm. incubations were done statically at 30°c. to determine the sulfate reduction activity, 0.5-ml sludge r was inoculated in duplo in 50 ml medium with 20 mm sulfate and different organic substrates. in time, samples were taken to determine the sulfide concentrations. controls were included without electron donor and without sulfate. conversion and product formation from citrate was determined using 0.05 ml of sludge r in 50 ml medium with and without sulfate. samples were taken in time and analyzed for organic acids. to get an impression of the abundance of different groups of bacteria, serial dilutions of suspensions of sludge r and sludge s were made in media with different substrates. the sludges were suspended by taking a sample by syringe and pressing the sample repeatedly back into the bottle with medium using needles with different sizes. the growth tests were done in 28-ml tubes. the incubation temperature was 30°c. isolation and characterization of strains strain r210 was isolated from the above enrichment of sludge r with 10 mm citrate without sulfate. strain s101 was isolated from the highest dilution with growth of sludge s in media with 10 mm citrate without sulfate. pure cultures were obtained by repeated dilution in citrate-media using, each time, the highest dilution with growth. these incubations were done in 28-ml tubes with 10 ml medium and 1 ml of different dilutions of inoculum. dilutions were made in tubes containing 9 ml medium without substrate or sulfate. purity was confirmed by microscopic observation after growth with different substrates. when grown with citrate, strain r210 and strain s101 formed typical crooked chains of cocci. when grown with sugars, strain r210 consisted of cocci and duplo-cocci. substrate tests were done in 28-ml tubes with 10 ml medium. growth substrates were tested at the indicated concentrations. growth was determined by the increase in optical density at 660 nm, substrate conversion, and product formation. electron acceptor utilization was tested by determination of product formation from citrate, and in the case of iron (iii), by color change of the medium. for phylogenetic analysis of the isolated strains, cells of 10-ml cultures were concentrated by centrifugation. genomic dna was extracted from the cells using the ultraclean soil dna extraction kit (mo bio laboratories, west carlsbad, ca, usa) according to the manufacturer's instructions. the nearly complete 16s ribosomal rna (rrna) gene was amplified using primers gm3f and gm4r (muyzer et al. 1995) . polymerase chain reaction products were purified and sequenced by a commercial company (baseclear, leiden, the netherlands). the sequences were first compared to sequences stored in the genbank using the blastn algorithm (http://www.ncbi.nlm.nih.gov/blast). subsequently, they were imported into the arb database (ludwig et al. 2004) , aligned, and added to a phylogenetic tree using the quick_add_to_existing_tree tool. the alignment was further corrected by eye, and a tree was calculated using the neighbor-joining algorithm with felsenstein correction. the rrna gene sequences of strain r210 and s101 are deposited in genbank/embl/ddbj under the accession numbers fj374769 and fj374768, respectively. organic acids were measured by high-performance liquid chromatography, and hydrogen and methane were measured by gas chromatography, as described previously (stams et al. 1993) . formate was analyzed colorimetrically using the method described by (lang and lang 1972) . sulfide was analyzed by the method of (trüper and schlegel 1964) . the vss content of the sludges was determined from the difference in dry weight (drying overnight at 105°c) and ash content (2 h at 600°c). the physical appearance of sludge samples at the influent point of the reactor and in the central part of the reactor suggested that citrate degradation and sulfate reduction were not directly coupled. when incubated unfed sludge r had a higher sulfate-reducing activity than sludge s (results not shown). the sulfate reduction activity of sludge r from the anaerobic reactor was determined with different substrates over a period of 3 days. the initial sulfide production activity of that sludge without added substrates was 18±3 mmol/g vss·day. in the absence of sulfate, no sulfide production was observed (<2.5 mmol/g vss·day). the following sulfide production activities (in mmol/g vss·day) were measured when substrates were added: citrate (59±9), formate (50±7), acetate (58±8), propionate (76± 21), butyrate (33±17), and lactate (116±22). the activity was highest with lactate, while the activity with butyrate was lowest. the effect of sulfate on citrate conversion by sludge r was determined. rapid growth and citrate conversion was observed both in the presence (not shown) and in the absence of sulfate (fig. 1) . in both cases, acetate and formate were formed as main products. the incubations were continued, and after 2 weeks, products were analyzed again. in the incubation without sulfate, still, 19.5 mm acetate was present, while in the incubation with sulfate, the acetate concentration had decreased to 8.5 mm. in this latter incubation, the sulfide concentration was about 18 mm (results not shown). serial dilutions of sludges r and s were made in media with citrate as substrate. sludge r showed growth with citrate till the 10 5 dilution, while sludge s showed growth till the 10 7 dilution. the vss content of sludge r and s was 3% and 0.2% (w/v), respectively. this shows that the relative number of citrate-degrading bacteria in sludge s is about 1,000 times higher than in sludge r. the cell numbers enumerated in the dilutions in media with and without sulfate were the same. the results that were obtained in these initial studies indicated that citrate was not a direct substrate for sulfate reducers, but that citrate was first fermented, and that the fermentation products were the substrates for the sulfate-reducing bacteria. as the sulfate-reducing bacteria of the inoculum were analyzed previously (dar et al. 2007 ), we focused our further research on bacteria responsible for citrate fermentation. upon microscopic observation of sludge r and sludge s and the enrichment of sludge r in media with citrate, coccoid cells were observed which formed twisted chains. two citrate-fermenting bacteria were isolated: strain r201 from sludge r and strain 101 from sludge s. the two strains were coccoid, but strain s101 was somewhat smaller in size than strain r201. both strain r210 and strain s101 degraded citrate mainly to acetate and formate (table 1 and table 2 ). strain s101 formed less formate than strain r210, but it formed some hydrogen. the substrate spectrum of the two strains was different. strain r210 was able to ferment sugars to acetate, formate, ethanol, and lactate. lactate was not degraded by this strain. by contrast, strain s101 did not ferment sugars but was able to ferment lactate and some other organic acids to acetate, formate, propionate, and hydrogen. taking into account the expected formation of bicarbonate, the carbon and electron balances fitted rather well with the expected balances. strain r210 and strain s101 did not show growth by respiration. both strains formed formate as a main product of citrate fermentation. however, strain s101 formed less formate in the presence of nitrate or crotonate. with these electron acceptors, about 4 mm formate was formed during growth with 10 mm citrate, while, in the controls, about 5.6 mm was formed. strain s101 was able to grow with fumarate. this compound was fermented to mainly formate, acetate, and propionate. when strain s101 was grown with a mixture of citrate and fumarate, the concentration of products was similar to the sum of products formed with citrate and fumarate, separately. oxygen was not used by the strains, but they could be grown (two transfers) in nitrogen-flushed media in which the reductant and sulfur source sodium sulfide was replaced by sodium sulfate. growth in sulfide-reduced media under air was possible but not when the cultures were shaken. comparative analysis of the 16s rrna gene sequences of the two strains showed that strain r210 was affiliated to members of the genus trichococcus, while strain s101 was affiliated to members of the genus veillonella (fig. 2) . strain r210 was closest related to trichococcus pasteurii (99.5% rrna gene sequence similarity). the closest relative of strain s101 was veillonella montepellierensis with an rrna gene sequence similarity of 96.7%. unfortunately, strain s101 was lost upon storage. strain r210 is deposited in the german collection of microorganisms and cell cultures (dsmz) as trichococcus sp. r210 (accession number dsm 22150). citrate is clearly not the direct substrate for the sulfatereducing bacteria in the bioreactor that was studied. recently, (gámez et al. 2008 ) found a rapid fermentation (antranikian and giffhorn 1987; bott 1997) . the two bacteria that we have isolated formed mainly acetate, formate, and presumably, bicarbonate from citrate. the two strains were able to ferment a set of others substrates as well. strain r210 fermented sugars. with these substrates, it formed, besides acetate and formate, also ethanol and lactate as products. strain s101 was not able to grow with sugars, but it was able to ferment some substrates, including lactate and malate, forming propionate as product. the products that are formed by the two strains are direct substrates for the sulfate-reducing bacteria that were previously detected in the sludge that was used to start up the bioreactor (dar et al. 2007 ). this may have been beneficial for the rapid start-up of the process. strain r210 was a trichococcus strain. (scheff et al. 1984 ) isolated a filamentous bacterium from bulking sludge. this bacterium was described as trichococcus flocculifomis and is able to grow with citrate, pyruvate, and a variety of sugars and polyols. other trichococcus species have been described like t. pasteurii (former lactosphaera pasteurii) and trichococcus palustris (former ruminococcus palustris; liu et al. 2002; janssen et al. 1995b; zhilina et al. 1995) . there are differences in the substrate spectrum of the different trichococcus species, but the pattern of fermentation products that we obtained with strain r210 for citrate fermentation and sugar fermentation is characteristic for trichococcus species. strain s101 was closest related to v. montepellierensis; the rrna gene sequence similarity was 96.7%. v. montepellierensis was isolated from clinical samples (jumas-bilak et al. 2004) . it is able to ferment lactate and succinate to propionate, while it does not ferment sugars. citrate was not tested as substrate of that strain. in general, members of the genus veillonella ferment lactate, but they are unable to ferment sugars. the substrate spectrum of strain s101 is similar as described for other veillonella species, though citrate utilization was rarely tested (kolenbrander 1992) . however, veillonella alcalescens is able to ferment citrate (de vries et al. 1977) . the substrate spectrum of the two citrate-fermenting bacteria is rather complementary. by the combined activity of these two bacteria, a wide variety of substrates is fermented to products that are excellent substrates for the sulfate-reducing bacteria present in the sludge. in this way, complex waste streams represent and excellent source of electron donors for sulfate reduction in industrial processes. interestingly, lactate can be fermented by strain s101 and can be degraded by a number of sulfate reducers. as shown by (laanbroek et al. 1983) , the lactate concentration and the prevailing conditions determine which of the bacteria preferentially degrades lactate. citrate metabolism in anaerobic bacteria anaerobic citrate metabolism and its regulation in enterobacteria sulfur and sulfate reduction with acetate and propionate in an aerobic biotechnological process for sulfide removal coexistence of physiologically similar sulfate reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor atp formation associated with fumarate and nitrate reduction in growing cultures of veillonella alcalescens anaerobic degradation of citrate under sulfate reducing and methanogenic conditions metabolism of sulfate-reducing prokaryotes new developments in reactor and process technology for sulfate reduction biological sulphide oxidation in a fed-batch reactor lactosphaera gen. nov., a new genus of lactic acid bacteria, and transfer of ruminococcus pasteurii schink 1984 to lactosphaera pasteurii comb. nov industrial applications of new sulphur biotechnology biological removal of sulfurous compounds from inorganic wastewaters veillonella montpellierensis sp. nov., a novel, anaerobic, gram-negative coccus isolated from human clinical samples the genus veillonella competition for l-lactate between desulfovibrio, veillonella, and acetobacterium species isolated from anaerobic intertidal sediments spezifische farbreaktion zum direkten nachweis der ameisensäure biotechnological treatment of sulfate-rich wastewaters emended description of the genus trichococcus, description of trichococcus collinsii sp. nov., and reclassification of lactosphaera pasteurii as trichococcus pasteurii comb. nov. and of ruminococcus palustris as trichococcus palustris comb. nov. in the low-g+c gram-positive bacteria reclassification of the sulfate-and nitrate-reducing bacterium desulfovibrio vulgaris subsp. oxamicus as desulfovibrio oxamicus sp. nov., comb. nov arb: a software environment for sequence data the ecology and biotechnology of sulphate-reducing bacteria phylogenetic relationships of thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16s rdna fragments sulfate reduction in methanogenic bioreactors desulfobacca acetoxidans gen. nov. sp. nov., a novel acetate-degrading sulfate reducer isolated from sulfidogenic sludge isolation of a new genus of sulfate-reducing bacteria and description of a new species of this genus, desulfomicrobium apsheronum gen trichococcus flocculiformis gen. nov. sp. nov. a new gram-positive filamentous bacterium isolated from bulking sludge growth of syntrophic propionate-oxidizing bacteria with fumarate in the absence of methanogenic bacteria metabolic interactions between methanogenic consortia and anaerobic respiring bacteria sulphur metabolism in thiorhodaceae. i. quantitative measurements on growing cells of chromatium okenii occurrence of methanogenesis during startup of a full-scale synthesis gas fed reactor treating sulfate and metal rich wastewater methanol conversion in high-rate anaerobic reactors thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor competition for h 2 between sulphate reducers, methanogens and homoacetogens in a gas-lift reactor dissimilatory sulfate-and sulfurreducing bacteria ruminococcus palustris sp acknowledgments we thank the technology foundation stw, applied science division of the netherlands science foundation nwo, chemical sciences (cw) of nwo, and the technology program economy ecology technology for financial support of our research.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012096-d7e89x03 authors: zhang, tong; ye, lin; tong, amy hin yan; shao, ming-fei; lok, si title: ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater treatment bioreactors date: 2011-06-25 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3408-y sha: doc_id: 12096 cord_uid: d7e89x03 in this study, dideoxy sequencing and 454 high-throughput sequencing were used to analyze diversities of the ammonia monooxygenase (amoa) genes and the 16s rrna genes of ammonia-oxidizing archaea (aoa) and ammonia-oxidizing bacteria (aob) in six municipal wastewater treatment plants. the results showed that aob amoa genes were quite diverse in different wastewater treatment plants while the 16s rrna genes were relatively conserved. based on the observed complexity of amoa and 16s rrna genes, most of the aob can be assigned to the nitrosomonas genus, with nitrosomonas ureae, nitrosomonas oligotropha, nitrosomonas marina, and nitrosomonas aestuarii being the four most dominant species. from the sequences of the aoa amoa genes, most aoa observed in this study belong to the cgi.1b group, i.e., the soil lineage. the aob amoa and 16s rrna genes were quantified by quantitative pcr and 454 high-throughput pyrosequencing, respectively. although the results from the two approaches show some disconcordance, they both indicated that the abundance of aob in activated sludge was very low. electronic supplementary material: the online version of this article (doi:10.1007/s00253-011-3408-y) contains supplementary material, which is available to authorized users. nitrification, the biological oxidation of ammonium to nitrite and nitrate, is an essential process in nitrogen cycling and wastewater treatment bioreactors. several groups of microorganisms are involved in the two-step process: ammonia-oxidizing archaea (aoa), ammonia-oxidizing bacteria (aob), and nitrite-oxidizing bacteria (nob). in the first step, aoa and/or aob oxidize nh 3 to no 2 − , and in the second step nob oxidize no 2 − to no 3 − . the first step is rate limiting and has been relatively well studied (limpiyakorn et al. 2006; park and noguera 2004; tokutomi et al. 2010) . although some heterotrophic bacteria (robertson and kuenen 1990) and anaerobic ammonia-oxidizing (anammox) bacteria (strous et al. 1999) can also oxidize ammonia to nitrite, aoa and aob are thought to be the main contributors for environmental ammonia oxidation and in bioreactors (nicol and schleper 2006) . according to the previous reports, nitrosomonas and nitrosospira are the most important genera of aob in activated sludge (park and noguera 2004; purkhold et al. 2000) . of the two genera, nitrosomonas has been shown to be the dominant aob in many bioreactors (limpiyakorn et al. 2006; park and noguera 2004; wells et al. 2009 ). by contrast, nitrosospira sp. were rarely found in activated sludge (schramm et al. 1999) , probably due to their relative low growth rate (siripong and rittmann 2007) resulting in an underrepresentation in bioreactors. the limited studies of aoa species in activated sludge showed a composition different from those existing in other environments such as soil, water column, and sediment (park et al. 2006) . moreover, their abundances seemed to be much lower (four orders or more) than aob based on analysis of amoa gene copy number (jin et al. 2010; limpiyakorn et al. 2010; wells et al. 2009 ). in this study, the aoa and aob diversity in six bioreactors from the wastewater treatment plants (wwtps) in four countries was investigated using amoa and 16s rrna genes as the biomarkers. high-throughput pyrosequencing of the 16s rrna genes was used to tabulate the aob diversity and their relative abundance in the total bacterial community. quantitative polymerase chain reaction (qpcr) was also used to quantify aob amoa genes in the same activated sludge samples. several sybr green-based qpcr systems have been tried to quantify aoa amoa gene. however, non-specific amplification and/or the formation of primer dimer hindered the accurate quantification of aoa amoa using this approach. in this study, activated sludge samples were taken from aeration tanks of eight full-scale wwtps treating municipal wastewater in china, singapore, canada, and the usa. relevant parameters about these wwtps are shown in supplementary table s1 . sludge samples from the aeration tank were fixed with 50% ethanol (v/v) on site before transporting to the laboratory for dna extraction. ten milliliters of fixed activated sludge samples was centrifuged at 4,000 rpm for 10 min at 4°c. approximately 200 mg of sample pellet was recovered for dna extraction in duplicate with a fastdna® spin kit for soil (qiagen, ca, usa), which was found to be the most suitable dna extraction method for the samples in this study, as being compared with other commercial reagents. primer set amoa-1f (5′-ggggtttctactggtggt-3′) and amoa-2r (5′-cccctckgsaaagccttcttc-3′) (rotthauwe et al. 1997 ) was used to amplify bacterial amoa gene in a 30-μl mixture containing 0.2 μl of takara ex taq tm , 3 μl of 10× ex taq buffer (takara), 3 μl of 10 mm dntp mixture (takara), 0.2 μm of each primer, and 20-50 ng of genomic dna. thermal cycling parameters followed the protocol of rotthauwe and colleagues (1997) . aob amoa gene copy numbers were quantified by using an icycler iq system (bio-rad, hercules, ca, usa) in triplicate with primer set amoa-1f/amoa-2r. quantitative real-time pcr amplification was performed in a total volume of 30 μl containing 15 μl of iq tm sybr ® green supermix (bio-rad), 5 μl of dna template with the concentration of about 1 ng/μl, and 0.3 μm of each primer using the same cycling conditions. the archaeal amoa gene was amplified by pcr using the primer set arch-amoaf (5′-staatggtctggcttagacg-3′) and arch-amoar (5′-gcggccatccatctgtatgt-3′) (francis et al. 2005) . pcr amplification was performed in a 50-μl volume comprising 25 μl 2× mightyamp buffer (takara), 1 μl (1.25 u) mightyamp dna polymerase (takara), 0.3 μm of each primer, and 20-50 ng of genomic dna. pcr was first incubated at 98°c for 1 min and was followed by 35 cycles at 98°c for 10 s, 60°c for 15 s, and 68°c for 60 s. the pcr products were visualized by agarose (1%) gel electrophoresis in the presence of suitable size markers. for high-throughput 454 pyrosequencing, the bacterial dna was amplified with a set of primers targeting the hypervariable v4 region of the 16s rrna gene. the forward primer is 5′-aytgggydtaaagng-3′ and the reverse primers are the mixture of four equally mixed primers: 5′-taccrgggthtctaatcc-3′, 5′-tacca-gagtatctaattc-3′, 5′-ctacdsrggtmtctaatc-3′, and 5′-tacnvgggtatctaatcc-3′ (rdp's pyrosequencing pipeline, http://pyro.cme.msu.edu/pyro/help.jsp). barcodes that allowed sample multiplexing during pyrosequencing were incorporated between the 454 adaptor a and the forward primer. dideoxy dna sequencing pcr products were purified by using pcrquick-spin tm pcr product purification kit (intron biotechnology, korea). the purified pcr products were ligated to pmd®18-t vector (takara). recombinant plasmid was transformed into e. coli and white colonies that grew on lb plates containing ampicillin (60 μg/ml), x-gal, and iptg were picked to conduct colony pcr amplification with the primer set m13f and m13r. the pcr products were purified and sequenced by abi 3730xl capillary sequencers (applied biosystems, foster city, ca, usa) with the primer m13f. an aob amoa gene clone library was constructed for each of the eight activated sludge samples (sample ids-cn-nj-sj, cn-hr-un, sg-sg-up, cn-qd-td, cn-sh-ts, cn-bj-jx, us-co-co, and ca-gp-gp). individual aoa amoa gene clone libraries were successfully constructed for samples cn-nj-sj, cn-hr-un, and sg-sg-up, while the other samples failed to generate library products. approximately 20 clones in each clone library were selected randomly and sequenced. the resulting aob amoa gene sequences were aligned and the jukes-cantor distances between subsequent pairs of sequences were calculated with dnadist from the phylip package (http://www.phylip.com/), and were grouped into 43 otus with a distance cut-off of 3%. in order to construct the phylogenetic tree, one sequence in each otu was selected, merged, and aligned with the reference sequences from ncbi entrez database. the neighbor-joining phylogenetic tree of aob amoa gene sequences was created by mega software (kumar et al. 2008) . the translated protein sequences were assigned to different otus with a 3% distance cut-off. the phylogenetic tree of aob amoa protein sequences was also created by mega software. aoa amoa gene and aoa amoa protein sequences were analyzed in the same manner as that of aob. the aob 16s rrna gene sequences were also classified into different otus with a 3% distance cut-off. in addition, good's estimator of coverage (good 1953) was calculated for each aoa and aob amoa gene clone library under 3% distance cut-off (table s2) . high-throughput pyrosequencing pcr amplicon libraries were prepared using a minimal number of amplification cycles (25 cycles) to minimize the accumulation of pcr artifacts. amplicons were purified using pcrquick-spin tm columns (intron biotechnology). equal amounts of amplicon products bearing individual sequence barcode for each sludge samples were combined for multiplex pyrosequencing on the roche 454 flx titanium platform (roche, nutley, nj, usa). following pyrosequencing, python scripts were written to (1) remove sequences containing more than one ambiguous base ('n'), (2) check the sequence integrity of the barcodes and partitioned the multiplex reads to the individual samples, and (3) remove sequence reads shorter than 150 bases. the resulting filtered reads were then compared with the greengenes 16s rrna gene database (desantis et al. 2006 ) using ncbi's blastn tool (altschul et al. 1990) with default parameters set to a maximum hit number of 100 (claesson et al. 2009 ). sequences were then assigned to ncbi taxonomies with megan (huson et al. 2007) using the lowest common ancestor (lca) algorithm and default parameters (absolute cut-off-blast bitscore 35; relative cut-off-10% of the top hits). the sequences obtained from clone library in this study were deposited in genbank under accession numbers jf271927-jf271985. the pyrosequencing results are deposited into the ncbi short reads archive database (accession number sra026842.2). diversity of aob amoa gene and 16s rrna gene in different activated sludge samples table s2 suggested that the coverage of the aob amoa gene clone libraries was over 70% except sample cn-sh-ts with a relatively low coverage (65%). as shown in fig. 1 , a total of 43 otus were generated based on 163 aob amoa gene sequences in eight clone libraries with a 3% distance cut-off. figure 1 reveals an interesting phenomenon that relatively few amoa otus were shared among the activated sludge samples, indicating that the amoa genes were quite diverse and not widely disseminated. figure 2 illustrated that most of these bacterial amoa otus were affiliated to nitrosomonas genus with nitrosomonas ureae, nitrosomonas oligotropha, nitrosomonas marina, and nitrosomonas aestuarii being the four most dominant species. however, only two otus (otu-17 and otu-32) were in the nitrosospira lineage and these two otus represented only eight sequences in the total 163 sequences, indicating the low abundance of nitrosospira in various types of activated sludge. figure s1 shows the relative abundance and distribution of aob amoa protein otus in different activated sludge samples. compared with aob amoa gene otus (shown in fig. 1 ), the diversity of amoa protein (a total of 26 otus) was less than that of amoa gene (43 otus) due to codon wobble positions. twelve otus of aob amoa protein were shared by at least two activated sludge samples. our findings indicate that a number of different amoa genotypes encode essentially the identical or very similar proteins possibly reflecting the functional constraints in efficient ammonia oxidation. as shown in the phylogenetic tree of aob amoa protein in fig. s2 , most otus were affiliated with n. ureae, n. oligotropha, n. marina, and n. aestuarii lineages, exactly the same as the amoa gene phylogenetic tree. there were only a few otus in nitrosospira lineage and n. europaea lineage, which was also identical with the above amoa gene phylogenetic analysis. in addition to the amoa gene, the 16s rrna gene was also used to investigate the aob community in six samples (i.e., cn-nj-sj, cn-qd-td, cn-hr-un, us-co-co, sg-sg-up, and ca-gp-gp) by high-throughput pyrosequencing. as shown in table 1 , more than 20,000 16s rrna gene fragment sequences were obtained for each sample site. these sequences were assigned to ncbi taxonomies using blast and megan software. a tabulation of aob-like sequences is shown in table 1 . the filtered reads were merged, aligned, and classified into 18 otus with a jukes-cantor distance cut-off of 3%. figure 3 shows that most (about 61%) aob 16s rrna otus were shared by more than two sample sites, and two major otus, i.e., otu-14 and otu-15, accounted for 49% of total sequences. these results implied the aob 16s rrna genes are much more conservative than amoa gene and amoa protein. along with direct tabulation of aob 16s rrna genes by pyrosequencing, we also quantify aob abundances in the activated sludge samples by qpcr, normalizing the aob amoa gene copy number against per nanogram of the genomic dna extracted from activated sludge. table 1 shows the abundances of aob amoa genes in different activated sludge samples quantified by qpcr. sample cn-sh-ts has the most abundant aob amoa gene copy numbers among the eight activated sludge samples tested. table 1 lists the total sequences obtained from pyrosequencing and the aoblike sequences in each of the six samples. aob sequences were successfully obtained in five out of the six samples, and the percentages range from 0.29% to 0.64%. for us-co-co, a sample from columbia, usa, no aob sequences were obtained, probably due to the low abundance of aob in this wastewater treatment plant, in agreement with amoa gene quantification results. in the present study, multiple experiments were attempted to amplify amoa gene from the eight samples using four pcr systems (including takara ex taq, takara mighty amp dna polymerase, sigma taq dna polymerase, and bio-rad sybr ® green supermix) at different thermal conditions (annealing temperature from 50 to 60°c). amplification was successful from only five samples as shown in fig. s3 and only three samples were eventually successfully cloned and sequenced. additionally, the presence of primer dimers was very serious in all samples other sequences were obtained from genbank. the tree was out-grouped with nitrosococcus halophilus (gammaproteobacterial aob) amoa sequence and it was not possible to quantify amoa using sybr ® green qpcr approach applied in this study (supporting information fig. s3 ). however, it is still of interest to have a preliminary assessment of the diversity of aoa amoa gene in the activated sludge samples by exploring the sequences obtained from the clone libraries of the samples cn-nj-sj, cn-hr-un, and sg-sg-up, all of which had good's estimator of coverage over 80% (table s2 ). the 56 sequences from the three samples were classified into 15 otus using a distance cut-off of 3% (fig. 4) . different from aob amoa otus (fig. 1) , some aoa amoa otus were shared among multiple samples. judging from the results in this study, it was found that the aoa amoa genes are not as diverse as aob amoa gene in different activated sludge samples. while it should be mentioned that the sample amount in this study was kind of limited, thus this issue needs more studies to be confirmed in the future. as shown in the phylogenetic tree of aoa amoa gene sequences in fig. 6 , most of the sequences obtained in this study were distinctly different from the previously reported sequences, especially the sequences recovered from marine and sediment. only two otus (otu-10 and otu-11) were grouped into the marine and sediment lineages. diversity of aob amoa gene and 16s rrna gene in different activated sludge samples a previous study reported that the amoa gene similarities among different aob species ranged from 65% to 100% (purkhold et al. 2000) . the similarities of the otus obtained in this study were 62% to 100%. the high diversity of the amoa gene found in this study was likely due to the different geographic locations of these sludge samples and the different operation/sewage conditions in these wwtps. the results in this study were consistent with previous reports that showed nitrosomonas instead of nitrosospira being the dominant aob in nitrification bioreactors in wwtp (park and noguera 2004; wells et al. 2009 ). notably, seven otu-17-associated sequences were all from the sample cn-hr-un taken from harbin, a city in northern china. the latitude of this city is the highest among those sampled in this study. our observation is consistent with the previous report that nitrosospira spp. prefer low temperatures of 4-10°c (avrahami et al. 2003; siripong and rittmann 2007) . we also observe a small number of otus belonging to nitrosomonas europaea lineage and several otus (otu-1, otu-3, otu-22, and otu-26) grouped far away from the known aob species. it was reported that the aob 16s rrna gene similarities generally ranged from 90% to 100%, much higher than those of amoa gene (about 65-100%) and amoa protein (about 72.5-100%) (purkhold et al. 2000) . the phylogenetic tree in fig. 5 shows that all of these sequences obtained in this study were grouped in nitrosomonas genus. there were 12 otus in nitrosomonas oligotropha group, four otus in the group of nitrosomonas ureae, and one otu in the n. marina and n. aestuarii lineages, indicating the dominance of these species in the activated sludge samples. this almost perfectly matches the above results of this study using amoa gene as the biomarker. a minor exception is that nitrospira 16s rrna gene was not found by pyrosequencing, probably due to the low abundance of these aob species in activated sludge and that the sequencing depth (>20,000 reads per sample) of the pyrosequencing applied in this study was not of sufficient depth to represent the complexity of the rare aob sequences. although we identified 495 sequence reads to the betaproteobacterial aob, no reads can be assigned to the gammaproteobacterial aob 16s rrna genes, indicating the dominance of betaproteobacterial aob in samples of this study. the abundances of aob in activated sludge were quantified using qpcr and pyrosequencing. however, the two data sets show a certain degree of discordance that might be due to the following reasons: (1) primer bias in qpcr amplification may introduce many uncertainties, especially when the target gene copy numbers are at low levels (herrmann et al. 2008; smith et al. 2006 ); (2) the 454 pyrosequencing conducted in this study may not be at a sufficient read depth to reflect the aob complexity since aob sequences was not detected in one of these samples; (3) the qpcr normalization step made use of total activated sludge dna, which contains nucleic acids from eukaryotes, bacteria, archaea, and viruses to which any variations in the relative distribution of these components between samples would distort the qpcr normalization process; and (4) 16s rrna gene copy numbers in prokaryotic microorganisms (klappenbach et al. [soil] [wwtp] [wwtp] [wwtp] [soil] [soil] [marine] [wwtp] [marine] [marine] [wwtp] [sediment] [sediment] [marine] [wwtp] [sediment] [sediment] [soil] [soil] [sediment] sequences recovered in this study are shown with "otu-" in the names. other sequences were obtained from genbank. the words in square brackets indicate the source the sequences originated from 2001) are different and the amoa gene copy numbers in different aob are also different (norton et al. 2002) . diversity of aoa amoa gene in different activated sludge samples although many reports described the diversity and abundance of aoas in the natural environments, only limited information is available describing aoa diversity in the activated sludge (limpiyakorn et al. 2010; park et al. 2006; wells et al. 2009; zhang et al. 2009 ). one possible reason for this situation could be due to the difficulty in pcr amplification of aoa amoa from dna of sludge samples. in this study, only five out of eight samples were successfully amplified, and only three samples were eventually successfully cloned and sequenced. previous studies revealed that most of aoa should be assigned to the clusters of cgi.1a (the marine and sediment lineage) and cgi.1b (the soil lineage) in the phylum crenarchaeota (hatzenpichler et al. 2008) . in this study, it was found that most of the aoa in the activated sludge samples have a closer distance to those aoa found in soil and thus belonged to the soil lineage. this result was in agreement with the previous studies on aoa communities in activated sludge (limpiyakorn et al. 2010; park et al. 2006) , except for a study on the activated sludge in bioreactors treating saline sewage (jin, et al. 2010 ) which had dominant aoa communities belonging to the marine and sediment lineage. aoa amoa protein sequences were classified into nine otus using 3% distance cut-off. figure s4 shows the relative abundance and distribution of aoa amoa protein otus in different activated sludge samples, indicating lower diversity compared with aoa amoa gene and that most of the aoa amoa protein otus were shared among the three samples. the neighbor-joining phylogenetic tree based on aoa amoa protein sequences in fig. s5 demonstrates the same pattern as shown by the aoa amoa gene tree (fig. 6) , that is, most of the otus in this study were grouped together and had a far distance from those sequences of marine and sediment. interestingly, there are two otus (otu-6 and otu-7 in fig. s5 ) closely related to the marine and sediment aoa lineage. the two otus were only detected in the sludge sg-sg-up, a sample from singapore, implying that the occurrence of these otus might be due to the geographic location of singapore, which is near the ocean. the other two samples were taken from cities far from the ocean and thus their aoa populations mainly come from soil. basic local alignment search tool effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine greengenes, a chimera-checked 16s rrna gene database and workbench compatible with arb ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean the population frequencies of species and the estimation of population parameters a moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring archaea dominate the ammonia-oxidizing community in the rhizosphere of the freshwater macrophyte littorella uniflora megan analysis of metagenomic data characterization and quantification of ammonia-oxidizing archaea (aoa) and bacteria (aob) in a nitrogen-removing reactor using t-rflp and qpcr rrndb: the ribosomal rna operon copy number database mega: a biologistcentric software for evolutionary analysis of dna and protein sequences quantification of ammoniaoxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance abundance of amoa genes of ammonia-oxidizing archaea and bacteria in activated sludge of full-scale wastewater treatment plants ammonia-oxidising crenarchaeota: important players in the nitrogen cycle? diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria evaluating the effect of dissolved oxygen on ammonia-oxidizing bacterial communities in activated sludge occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors phylogeny of all recognized species of ammonia oxidizers based on comparative 16s rrna and amoa sequence analysis: implications for molecular diversity surveys combined heterotrophic nitrification and aerobic denitrification in thiosphaera pantotropha and other bacteria the ammonia monooxygenase structural gene amoa as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations microscale distribution of populations and activities of nitrosospira and nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants evaluation of quantitative polymerase chain reaction based approaches for determining gene copy and gene transcript numbers in environmental samples missing lithotroph identified as new planctomycete a novel control method for nitritation: the domination of ammonia oxidizing bacteria by high concentrations of inorganic carbon in an airliftfluidized bed reactor ammonia-oxidizing communities in a highly aerated full-scale activated sludge bioreactor: betaproteobacterial dynamics and low relative abundance of crenarchaea occurrence of ammonia-oxidizing archaea (aoa) in activated sludges of a laboratory scale reactor and two wastewater treatment plants acknowledgments the authors wish to thank the hong kong general research fund (hku7197/08e) for the financial support of this study. lin ye wishes to thank hku for the postgraduate studentship. we would also like to thank w. chan and c. k. wong for technical help in pyrosequencing.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012448-9cuq0jqg authors: roosen, christoph; müller, pia; greiner, lasse title: ionic liquids in biotechnology: applications and perspectives for biotransformations date: 2008-12-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1730-9 sha: doc_id: 12448 cord_uid: 9cuq0jqg ionic liquids are considered as an alternative to organic solvents for catalysis. the literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. the overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. it is apparent that advantages can be gained in view of activity, stability and selectivity. furthermore, integration of reaction and separation has a high potential in the field. the review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature. electronic supplementary material: the online version of this article (doi:10.1007/s00253-008-1730-9) contains supplementary material, which is available to authorized users. a pure salt which is liquid below a threshold temperature (usually 100°c) is denoted as an ionic liquid (il). the use of il is of high current interest for catalysis (parvulescu and hardacre 2007) , biocatalysis and other applications are emerging. ils are often discussed as the alternative to organic solvents in view of sustainable and ultimately "green" processes. some aspects of relevance for biocatalysis have not been reviewed critically so far: what advantages can be expected for biotransformations and catalysis? does the current state of research substantiate the rise of big expectations for the application of il in biocatalysis? is the basis for technical application substantiated by enzyme performance and/or productivities? as metrics for technical applicability and comparison we have calculated the productivity per mass of enzyme preparation and space-time yields (sty) from the references whenever possible (for tables see the supporting information). the results vary more than three orders of magnitude and, therefore, allow a semi-quantitative comparison. selected examples in view of technical applicability are given in more detail. the definition of il matches a broad variety of substances which not only in view of biocatalysis have little in common. the number of il and their properties are at least as wide as the broad variety of solvents available today. an even qualitatively applicable structure-properties relationship does not exist so far (bhargava et al. 2008) . in particular, the miscibility of il varies from virtually immiscible to fully miscible with water illustrating the range of different properties in view of biocatalysis. interaction with the (bio-)catalyst may lead to higher selectivities and stabilities enhancing productivities of the catalytic system. this has been shown for a broad number of biocatalysts and is known for conventional solvents as well. the present studies do support that the properties of il can provide gains for activity, stability and separation as properties can be varied widely. especially, separation can be facilitated by the inherently low vapor pressure of il allowing for easy removal of volatile substances. this can be carried out by sublimation, distillation or extraction with supercritical fluids in which il are virtually insoluble. the separation can be combined directly with the reaction in either continuous or batchwise applications. here, il can show selectivities which are difficult to obtain otherwise (eckstein et al. 2006; peters et al. 2007 ). however, the overall mapping of the biocatalysis in il (fig. 1) shows that the number of studies and their experimental approaches are not sufficient to support understanding of the influence of il on even the most prominent biocatalysts used (see below). so far, the early claims of generally favorable ecotoxicity and biodegradability of il investigated is not substantiated and is reduced to the negligible low vapor pressure compared to organic solvents, and some authors postpone this aspect to further developments . of specific relevance are purity, water activity, analysis of il as well as solutes of il which are still not resolved generally. particularly, trace impurities are known to have significant effects on (bio-)catalyst activity and stability (park and kazlauskas 2001) . therefore, synthesis related impurities as halide or silver ions are important and sometimes lead to poor reproducibility especially in early results in the field. this is addressed by either downstream purification (nockemann et al. 2005) or optimised continuous synthesis (minnich et al. 2007 ). as recognized early on for conventional solvents water is of importance for both activity and stability of biocatalysts. here, the concept of water activity a w as opposed to water content is of importance to gain basic understanding and to distinguish between effects from the solvents and the availability of water (eckstein et al. 2006) . furthermore, the analysis of il and solutes is no routine work and care has to be taken to obtain valid results (baker et al. 2005) . the cations and anions in il applied for biocatalysis found in the open literature are shown in figs. 2 and 3, respectively. from the 20 cations and 26 anions yielding 520 combinations, only 11% have been applied so far; not taking into account cation and anion which have been shown to be il but have to the best of our knowledge not been applied to biocatalysis. therefore, the exploration map of biocatalysts probed in il remains sketchy even more as the approaches taken by various groups render direct comparison difficult (fig. 1) . expectably, the emphasis of the research carried out lies on commercially available il. bmim-based il have gained the main attention with bf 4 and pf 6 and recently ntf 2 and otf as anions (see fig. 2 and 3 for structures and abbreviations). as biocatalysts, mainly hydrolases (enzyme class 3) have been employed with candida antarctica lipase b (calb) in free form or an immobilised preparation accounting for more than 75% of the contributions to the field. surprisingly, the role of enzyme preparation on performance is generally not addressed even though ph memory and additives are known to dominate activity and stability in conventional solvents when an insoluble enzyme is applied (gupta 1992; klibanov 1987) . the application of il in monophasic systems is either as pure solvents or as miscible additives to aqueous buffer systems. for the use as pure solvent, only a few studies can be found that allow comparison on the basis of a w . for the kinetic resolution of (r,s)-1-phenylethanol via transesterification with vinyl acetate catalysed by pseudomonas sp. lipase (chirazyme l-6) enantiomeric excess increased whereas conversion decreased with higher water activity for both methyl tert-butyl ether (mtbe) and [bmim][ntf 2 ] (eckstein et al. 2002) . at the same initial a w the catalytic activity was always higher in mtbe than in the il, whereas selectivity was higher in the il comparing the same level of conversion. in mtbe, the enzyme selectivity decreased fast above 50°c from an e value of 270 to 4 whereas in the il no loss of enantioselectivity was apparent for temperatures up to 70°c at e=150. the authors attribute the difference to the boiling point of either mtbe or of the reactants in the il with the lowest boiling point (vinyl acetate). stabilisation of insoluble enzyme preparations by il was reported by various groups. however, the majority of the reports are singular reports on enzymes and/or il comparing only few conventional solvents. the earliest report was on thermolysin to be stabilised in [bmim][pf 6 ] compared to ethyl acetate (erbeldinger et al. 2000) . furthermore, only a few studies take the effect of the enzyme preparation into account which has an impact as shown by the stabilisation of enzymes in a range of il with cross linked enzyme aggregates (clea; toral et al. 2007 (lozano et al. 2001 ). the reason is obscured by the wide variation of a w for the il at the chosen constant water content of 2% (v/v). interestingly, stability in il generally increased in the presence of dissolved substrates to less than 5% loss of activity within 4 h. increased stability was reported for bacillus stearothermophilus esterase immobilised on celite in [bmim][pf 6 ] compared to mtbe and hexane both at a w =0.11; half life in mtbe and hexane was determined with 80 h and 8 h, respectively, no inactivation in il was observed within 240 h (persson and bornscheuer 2005) . the authors attribute the stabilisation to electrostatic interactions between the il and the enzyme. candida rugosa lipase (crl) was recycled five times in [bmim] [pf 6 ], [omim] [pf 6 ], as well as in organic solvents (toluene and hexane) with decreases in activity of 5%, 8% and 50%, respectively, and a decrease of 15% for the enantioselectivity in il as compared to more than 50% for both tested organic solvents (persson and bornscheuer 2005) . later, storage (erbeldinger et al. 2000) . calb was found active only in [mtea] [meso 4 ] which was confirmed by minimal changes in the infrared spectrum of the dissolved enzyme (rantwijk et al. 2006) . the role of trace impurities in il was shown by using purified and non-purified [pmim] [bf 4 ] for acylation with pseudomonas cepacia lipase (park and kazlauskas 2001) . after purification activity is comparable to toluene (productivity 4.9 g g −1 in [bmim][pf 6 ] and 3.6 g g −1 in toluene) whereas the non-purified il completely inhibited the lipase. the activity loss is mainly attributed to silver ions which are introduced by some synthesis protocols. the activity and selectivity of calb in 19 il and hexane where compared for the transesterification of vinyl butyrate with 1-butanol (de los riòs et al. 2007) . a selectivity of 75% with sty of 11.2 g l −1 h −1 was found for hexane. in ten water-miscible il selectivities greater than 99% with sty at least one order of magnitude lower were reported with the exception of [bmim] [dca] at sty=18.2 g l −1 h −1 . the water-immiscible il showed lower selectivity of 79%-94%. however, the sty were generally higher (up to 170 g l −1 h −1 in [omim][pf 6 ]) than in the water-immiscible ones. as mentioned above the comparability may be questioned due to the experimental approach of constant water content of 2 vol.%. the potential of il as polar solvent for biocatalysis is demonstrated for polar substrates showing low solubility in enzyme compatible organic solvents. the acylation of the highly polar β-d-glucose catalysed by calb is a model reaction. the comparably high solubility of the substrate allows for high productivity and space-time yield (sty) in il (3.4 g g −1 and 2.84 g l −1 h −1 in [moemim] [bf 4 ]) in which the highest regioselectivity (93% at 99% conversion) was observed (itoh et al. 2001) . upon recycling of the enzyme-il mixture by extraction with hexane for four times the enantioselectivity remained at >99% but activity decreased 30-fold. the regioselective acylation of β-d-glucose with calb (novozym sp435) in two different organic solvents (acetone, thf) and eight il was carried out (park and kazlauskas 2001 a number of investigations shows that il as additive or solubiliser for aqueous systems can influence activity and selectivity and allows the conversion of otherwise insoluble substrates. comparing seven water-miscible mim-based il in different aqueous concentrations did also influence the selectivity of novozym 435 catalysed hydrolysis of d,lphenylglycine methyl ester (lou et al. 2006 ) reported on the stereoselective reduction of 2octanone by e. coli alcohol dehydrogenase 'a' in ilaqueous buffer mixtures. it was shown that an increase in the il to buffer ratio leads to decrease in enzyme activity. since these mixed phases allow for higher substrate concentrations, the maximum sty of the reaction could be increased fourfold to 2.2 g l −1 h −1 at a 2-octanone concentration of 1.2 mol l −1 with 90%(v/v) il. however, in another report, the same authors report similar results by using micro-aqueous-organic solvent mixtures (de gonzalo et al. 2007b ). the hydrolysis of n-acetyl amino acid ethyl esters by novo alcalase from bacillus licheniforms (mainly subtilisin carlsberg) in [etpy] [tfa]/water and acetonitrile/water (both 15%(v/v)) was compared (zhao and malhotra 2002) . all reported substrates were converted in the il system whereas in acetonitrile/water mixture, no activity was observed. for other substrates enantioselectivity and activity were comparable in solvents or better in the aqueous il system. in 2008, kragl and co-workers described the use of [mdepega]cl for the purification of two adh from lactobacillus brevis and a thermophilic bacterium (t-adh), respectively (dryer and kragl 2008) the il had a stabilizing effect on both enzymes during purification and for the t-adh during the reduction of acetophenone. furthermore, the il acts as a solubiliser for the low water-soluble substrate. the enantiomeric excess was unchanged at >99%; sty was increased by a factor of 2.5 when using 10% (w/w) il. with increased substrate concentration from 10 mmol l −1 to 60 mmol l −1 sty was increased by a factor of 1.7. enantioselectivity of the hydrolysis of butyl 2-(4-chlorophenoxy) propionate catalysed by the candida rugosa lipase could be increased by the addition of il to an aqueous buffer solution (mohile et al. 2004) . increasing the il to 50% (v/v) raised the ee from 47% to >99%. however, activity at these conditions was at least 12-fold decreased. product removal by distillation is facilitated by the negligible vapor pressure of the majority of il; by this means, recycling of the enzyme in the il is possible to some extent. five il were tested at initial a w =0.2 for the kinetic resolution of indinavir precursor by candida antarctica lipase b (lourenco et al. 2007) . in [bmim] [dca], the reaction proceeded without enantioselectivity but the highest activities were obtained in [mdepeg] [dca] with up to 98% enantiomeric excess. the isolation of the product was simplified as it can be removed and recovered under reduced pressure and the il could be reused once. for the transesterification of 1-phenylethanol with vinyl acetate, eight different enzymes in ten different il were investigated (schöfer et al. 2001) . for all but one lipase, best results were obtained in il. productivities up to 6.5 g g −1 and sty of 0.23 g l −1 h −1 were achieved. for calb in [bmim][ntf 2 ], the reuse of il and lipase for three times with no apparent loss of selectivity and 10% loss per cycle in activity was demonstrated. the removal of the products was feasible by distillation under reduced pressure. biphasic reaction systems facilitate the combination of reaction and separation. for biocatalysis the scope of substrates is broadened and by the proper choice of reactive and non-reactive phase thermodynamic boundaries can be maximised (eckstein et al. 2006; peters et al. 2007 ). unsurprisingly, numerous enzymatic reactions have been performed in biphasic systems with some examples for the beneficial application of il. they are either employed in aqueous biphasic systems as non-reactive alternatives to non-miscible organic solvents, or as immobilisation matrix and extracted by organic solvents or supercritical fluids (scf). whole cells of rhodococcus r312 were compared for the transformation of 1,3-dicyanobenzene to 3-cyanobenzamide in water/toluene and in water/[bmim][pf 6 ] (cull et al. 2000) . maximum conversion (44%) and selectivity was found to be higher in the il system. the initial activity was found to be ten-fold higher than that in the toluene system with no apparent loss within 90 min in the [bmim][pf 6 ] system, whereas in the toluene system residual activity increased six-fold within the observation period. the authors do not quantify the contribution of phase transfer kinetics or aggregation phenomena. the synthesis of isoamyl acetate with novozym 435 suspended in il and isoamyl alcohol/acetic acid/toluene/ water mixture as the non-reactive phase was compared for six il of which three are non-miscible with the non-reactive phase (fehér et al. 2008 [pf 6 ] of which the first was chosen for cost considerations. the reactive phase was reused seven times without apparent loss of activity; however, 21% mass loss of il is reported. the reaction was scaled up by a factor of 6.5 where the enzyme amount was increased by a factor of 32.5. here, the il phase was reused ten times. the highest productivities of 23 g g (biocatalyst) −1 (see supporting information) were obtained for the single-batch experiments. the hydrolysis of 3,4,6-tri-o-acetyl-d-glucal catalysed by celite-supported pseudomonas cepacia lipase was compared for the biphasic mixtures of buffer and [bmim] [pf 6 ] or [bmim] [bf 4 ] and monophasic thf/buffer (mohile et al. 2004 ). the regioselectivity was improved to 98% from 62% with comparable productivities for thf and [bmim] [pf 6 ]. alcohol dehydrogenase from lactobacillus brevis (lb-adh) was employed in biphasic systems consisting of an aqueous phase and [bmim][ntf 2 ] or methyl tert-butyl ether (mtbe) (eckstein et al. 2004 (eckstein et al. , 2006 . the reduction of 2-octanone to (r)-2-octanol using substrate-coupled regeneration of the cofactor nadph was performed with ee >99% and excellent selectivity in both systems. the partition coefficients of the co-substrate 2-propanol and the co-product acetone change from ∼1 in the mtbe system to 0.4 for 2-propanol and 2.0 for acetone in the il system thereby the limiting conversion is increased (eckstein et al. 2006) . a conversion of 88% is obtained within the 3 h compared to 61% in the system with mtbe. unfortunately, stability of the enzyme is higher in the presence of mtbe with a half life of >1,000 h as compared to the il with 50 h. the combination of il with scf is particularly useful as the il is not soluble in scf. therefore, the loss of il is negligible. in the case of carbon dioxide as scf, the usually high solubility in il with mole fractions of more than 0.6 and the viscosity of the il is decreased significantly (aki et al. 2004) . the combination of il as reactive phase and scf as non-reactive phase has been successfully applied to a range of catalytic systems. so far, only lipases have been employed as biocatalysts with calb in various il/carbon dioxide systems. this is probably due to the high stability of calb towards supercritical carbon dioxide (karmee et al. 2008) . calb was suspended in il on two different supports and was used in 4 h reaction-extraction cycles with scco 2 as non-reactive phase and stored for 20 h at room temperature (lozano et al. 2002) . the synthesis of butyl butyrate from vinyl butyrate and 1-butanol was most successful using [bmim] [ntf 2 ]. the reaction resulted in selectivity >95% and conversion >50% and exceptional sty and productivity of 11 kg g(enzyme) −1 . at 40°c, no deactivation after 32 h reaction time is apparent. successively, five ammonium-based il were tested for the continuous synthesis of butyl butyrate with scco 2 . partly purified novozym 525 l was imposed to silica gel and successively mixed with il. best results were obtained for the use of [c 4 tma][ntf 2 ] with reported synthetic yield of 59% and selectivity of 98.5% at comparable sty and productivity of 0.6 kg g(enzyme) −1 . over reaction, time yield and selectivity increased slightly. the authors assume the water content as the dominating factor. activity and stability tests showed that in all il the enzyme was more active and more stable than in hexane. in il no apparent deactivation was found. the kinetic resolution of secondary alcohols in the system il/scco 2 was target of investigations of reetz et al. (2002 reetz et al. ( , 2003 . for the batchwise reactions, rac-2-octanol and vinyl acetate were used as standard substrates in three different il (anion: bmim, cations: pf 6 , bf 4 , ntf 2 ) and 3 different preparations of calb (lyophilisate, novozym 435, sol-gel immobilisate). highest activity was obtained with the lyophilized preparation. comparing il best results were obtained with lipophilic non-coordinating anions. [ntf 2 ] led to the highest selectivity with quantitative selectivity, regardless of enzyme preparation. noteworthy, enzyme activity was not altered in il compared to isooctane. further secondary alcohols were tested for the batchwise kinetic resolution using novozym 435 and [bmim] [ntf 2 ]. different pressures and temperatures were applied for the extraction with scco 2 . selectivities around 95% could be reached. the mixture of il and immobilized enzyme were able to be recycled several times without apparent loss of activity and selectivity. for the continuous process, transesterification of rac-1-phenylethanol with vinyl laureate novozym 435 was suspended in [bmim] [pf 6 ]. the reaction was continuously operated for 112 h. the average yield of the (s)-acylation product was 81% with selectivity close to unity. the il-enzyme mixture was recycled with identical selectivity and activity proving the high long-term stability of the immobilized calb under given process conditions. follow-up work showed that similar results can be obtained using peg instead of il [lit.]. in 2002, iborra et al. (lozano et al. 2002) [ntf 2 ] as reactive phase for the continuous kinetic resolution of rac-1-phenylethanol with calb. the enzyme was imposed to celite, suspended in il, and used in 4 h reaction-extraction cycles followed by 20 h of storage at room temperature. as non-reactive phase again scco 2 was used. depending on temperature, selectivities in the range of 78% to 97% with conversions of >35% were obtained. good productivity and fair sty characterize the reaction. this was extended to the continuous dynamic kinetic resolution of rac-1-phenylethanol in the system il/scco 2 (lozano et al. 2006) . novozym 435 was combined with silica modified with benzenesulfonic acid (scx) as racemisation catalyst. both the chemical catalyst and the immobilized calb were mixed with il. as the mixture of both catalysts did not show any activity, they were physically separated by glasswool. in the plug-flow reactor, two layers of enzyme with one layer of scx in between were used. the reactor was continuously operated for 5 h. in both [emim][ntf 2 ] and [btma][ntf 2 ], the yield of the (r)-ester product reached its maximum of 76% compared to the maximum 50% in [bmim][pf 6 ] without racemisation catalyst. the coating of the scx with il enhanced selectivity. although the combined catalysts showed higher activity and selectivity in il than in hexane, only low sty and productivity could be observed in the system il/scco 2 . by using a different immobilisation technique using purified novozym 525 l on modified silica-c4 a yield of 78% of the desired (r)product (ee=92%) was observed with a more than 20-fold increase in sty=18 g l −1 h −1 and productivity of 1.7 g g (biocatalyst) −1 . in conclusion, ils extend the solvent range for biocatalysis in biphasic as well as monophasic systems. the examples highlight a potential in all relevant aspects of applications. il may have significant impact on selectivity, stability and activity. furthermore, no general trends can be identified for the interaction of biocatalysts and il. here, the database under experimentally comparable conditions has to be broadened considerably. additionally, the peculiar role of water for biocatalysis has to be recognised and experimentally implemented. the identification and control of impurities is an aspect which is recognised generally. especially the combination of reaction and separation will be of interest in the future as here the potential of il can be exploited to full extent. highpressure phase behavior of carbon dioxide with imidazoliumbased ionic liquids an analytical view of il modelling room temperature ionic liquids room-temperature il as replacements for organic solvents in multiphase bioprocess operations asymmetric biocatalytic reduction of ketones using hydroxyfunctionalised water-miscible il as solvents enzymatic reduction of ketones in "micro-aqueous" media catalyzed by adh-a from rhodococcus ruber the effect of il media on activity, selectivity and stability of candida antarctica lipase b in transesterification reactions ionic liquids for aqueous two-phase extraction and stabilization of enzymes at low water activity/alpha-chymotrypsin is more active in an il than in nonionic organic solvents use of an il in a twophase system to improve an alcohol dehydrogenase catalysed reduction maximise equilibrium conversion in biphasic catalysed reactions: mathematical description and practical guideline enzymatic catalysis of formation of z-aspartame in il-an alternative to enzymatic catalysis in organic solvents enzymatic production of isoamyl acetate in an ionic liquid-alcohol biphasic system optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-buoh enzyme function in organic solvents lipase-catalyzed enantioselective acylation in the il solvent system: reaction of enzyme anchored to the solvent technical aspects of biocatalysis in non-co 2 -based supercritical fluids enzymatic selective acylation of glycosides in il: significantly enhanced reactivity and regioselectivity enzymes that work in organic solvents efficient enantioselective hydrolysis of d,l-phenylglycine methyl ester catalyzed by immobilized candida antarctica lipase b in il containing systems enzymatic resolution of indinavir precursor in il with reuse of biocatalyst and media by product sublimation overstabilization of candida antarctica lipase b by il in ester synthesis continuous green biocatalytic processes using il and supercritical carbon dioxide chemoenzymatic dynamic kinetic resolution of rac-1-phenylethanol in il and il/supercritical carbon dioxide systems combining reaction calorimetry and atr-ir spectroscopy for the operando monitoring of il synthesis il: efficient additives for candida rugosa lipase-catalysed enantioselective hydrolysis of butyl 2-(4-chlorophenoxy) propionate purification of imidazolium ionic liquids for spectroscopic applications il as a new reaction medium for oxidase-peroxidase-catalyzed sulfoxidation improved preparation and use of roomtemperature il in lipase-catalyzed enantio-and regioselective acylations catalysis in il increased stability of an esterase from bacillus stearothermophilus in il as compared to organic solvents exploring conversion of biphasic catalytic reactions: analytical solution and parameter study biocatalysis in il structure and activity of candida antarctica lipase b in il biocatalysis in il: batchwise and continuous flow processes using supercritical carbon dioxide as the mobile phase continuous flow enzymatic kinetic resolution and enantiomer separation using il/supercritical carbon dioxide media enzyme catalysis in il: lipase catalysed kinetic resolution of 1-phenylethanol with improved enantioselectivity cross-linked candida antarctica lipase b is active in denaturing il enzymatic resolution of amino acid esters using il n-ethyl pyridinium trifluoroacetate acknowledgements for financial support, we thank the "arbeitsgemeinschaft industrieller forschungsvereinigungen" aif (kf0174001ul5), the ministry of education and research (bmbf, 17 55x 06) and deutsche forschungsgemeinschaft graduiertenkolleg 1166 (bionoco). for helpful discussion we thank prof. walter leitner (rwth aachen university).open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012432-te3sysra authors: chmura, a.; shapovalova, a. a.; van pelt, s.; van rantwijk, f.; tourova, t. p.; muyzer, g.; sorokin, d. yu. title: utilization of arylaliphatic nitriles by haloalkaliphilic halomonas nitrilicus sp. nov. isolated from soda soils date: 2008-11-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1685-x sha: doc_id: 12432 cord_uid: te3sysra an enrichment culture from saline soda soils, using acetate as carbon and energy source and 2-phenylpropionitrile as nitrogen source (ppn) at ph 10, resulted in the isolation of strain anl-αch3. the strain was identified as a representative of the genus halomonas in the gammaproteobacteria. the bacterium was capable of ppn utilization as a nitrogen source only, while phenylacetonitrile (pan) served both as carbon, energy and nitrogen source. this capacity was not described previously for any other haloalkaliphilic bacteria. apart from the nitriles mentioned above, resting cells of anl-αch3 also hydrolyzed mandelonitrile, benzonitrile, acrylonitrile, and phenylglycinonitrile, presumably using nitrilase pathway. neither nitrile hydratase nor amidase activity was detected. the isolate showed a capacity to grow with benzoate and salicylate as carbon and energy source and demonstrated the ability to completely mineralize pan. these clearly indicated a potential to catabolize aromatic compounds. on the basis of unique phenotype and distinct phylogeny, strain anl-αch3 is proposed as a novel species of the genus halomonas—halomonas nitrilicus sp. nov. electronic supplementary material: the online version of this article (doi:10.1007/s00253-008-1685-x) contains supplementary material, which is available to authorized users. nitriles are mostly synthetic compounds containing a c≡n functionality, although a few examples exist of naturally occurring nitriles formed by cyanogenic plants from cyanide (vetter 2000) or during anaerobic degradation of amino acids by bacteria (harper and gibbs 1979) . nitrile compounds are widely used as intermediates and building blocks in organic synthesis. for example, the enzymatic hydrolysis of racemic nitriles into enantiomerically pure αsubstituted carboxylic acids is of great industrial interest. nitriles can be used as precursors in the synthesis of αhydroxycarboxylic acids (breuer et al. 2004 ) and amino acids, which are pharmaceutical intermediates and important resolution agents in organic resolution processes (gröger 2001; ravichandran et al. 1998) . most nitriles are toxic and difficult to degrade. nevertheless, the potential for enzymatic hydrolysis of the nitrile bond is widely spread among bacteria and fungi (banerjee et al. 2002) . two different enzymatic systems are known to convert nitriles into the corresponding carboxylic acids. the metalloenzyme nitrile hydratase catalyzes the hydration of a wide range of aliphatic, arylaliphatic, and aromatic nitriles into the corresponding amides, which are then further converted into carboxylic acids and ammonium by amidases (kobayashi and shimizu 1998) . nitrilases hydrolyze nitriles, mostly aromatic, directly into acids and ammonium in a single step (kobayashi and shimizu 2000) . the microorganisms possessing these enzymes are valuable biocatalysts and can be used either in (enantioselective) organic synthesis or in environmental biotechnology (layh et al. 1997; bunch 1998; håkansson et al. 2005) . until now, all known nitrile-degrading microorganisms included only neutrophilic species, i.e., growing optimally at neutral ph values. however, a recent survey of naturally occurring haloalkaline habitats, such as soda lakes and soda soils, demonstrated the possibility of biodegradation of simple aliphatic nitriles at extremely alkaline conditions. the compounds, such as aceto-, propio-, butyro-, iso-butyro-, and valero-nitriles, were utilized as carbon, energy, and nitrogen source by a novel haloalkaliphilic gammaproteobacterium, an actinobacterium and bacilli (sorokin et al. 2007a, b) . the potential for enzymatic nitrile hydrolysis at highly alkaline conditions might have certain advantages, particularly when cyanide is involved in the reaction process. for example, a well-known chemical strecker reaction could be coupled with enzymatic α-aminonitrile hydrolysis to (enantioselectively) produce α-aminoamides and/or α-aminoacids (duthaler 1994) . in this paper, the biodegradation of a variety of arylaliphatic nitriles is demonstrated under extremely haloalkaline conditions by a novel species of the genus halomonas isolated from soda soils. the capacity to hydrolyze nitrile compounds has never been demonstrated previously for this metabolically diverse group of halo (alkali)philic gammaproteobacteria. a mixed sediment sample from kulunda steppe (southwestern siberia, altai, russia) soda lakes and a mix sample from soda solonchak soils (kulunda steppe and northeastern mongolia) were used as the inoculum for the enrichments. the ph and salinity of the lake brines were within the range of 9.5-11.05 and 50-400 g l −1 , respectively. the ph of water extract from the solonchak soil samples varied from 9.5 to 10.8 and the total salt content-from 30 to 200 g kg −1 . a mineral medium based on sodium carbonate buffer (ph 10, 0.6 m total na + ) was used for enrichments and pure culture study (g l −1 ): na 2 co 3 , 22; nahco 3 , 8; nacl, 6; k 2 hpo 4 , 0.5. the ph of this medium was stable even after prolonged incubation. after sterilization, the medium was supplemented with 1 ml trace metal solution l −1 (pfennig and lippert 1966) , 1 mm mgso 4 , and 0.1 mg l −1 of filter-sterilized vitamin b 12 . the enrichment was performed in 100-ml serum bottles closed with grey rubber septa (to prevent loss of substrate) containing 20 ml medium and 1 cm 3 sediment or soil mixture. nitriles were added directly from the pure source into the medium (2-4 mm) and the bottles were immediately sealed to prevent the substrate loss. it is necessary to stress that the nitriles used in the growth experiments and ph profiling (phenylpropionitrile (ppn) and phenylacetonitrile (pan)) were chemically stable up to ph 11 as was demonstrated in abiotic controls. in case of solid medium (solidifying agent was noble agar, difco), ppn was added after cooling the medium down to 50°c to prevent excessive loss of substrate and the plates were incubated in closed jars for 30 days. liquid cultures were incubated on a rotary shaker at 150 rpm and 28°c and were periodically checked for growth and ammonia formation. the positive cultures were serially diluted and the culture obtained from a maximal positive dilution was plated onto the solid medium. separate colonies were placed into liquid medium with ppn and acetate in 30-ml serum bottles closed with rubber septa and with 5 ml liquid. positive cultures were plated again to check for the purity. growth experiments with pure cultures were performed in 1 l closed serum bottles with 100 ml liquid on a rotary shaker at 150 rpm and 30°c. substrates were used at 2-20 mm concentration. growth was monitored by optical density and the degradation of nitriles was followed by ammonium production, disappearance of substrate, and formation of intermediates. the ph profiling of growth was performed in the medium containing 0.6 m total na + either as nacl (ph 6.5-8.0) or nahco 3 /na 2 co 3 (ph 8.5-11.0). the salt dependence of growth was investigated in a range of sodium carbonate-based media containing 0.1-4.0 m total na + at ph 10. the substrate utilization spectrum was investigated in standard medium at ph 10 using nh 4 cl (4 mm) as the nitrogen source. to determine the spectrum of nitrile degradation and the influence of ph and salt concentration on the nitrilase activity, the cells grown at ph 10 and 0.6 m na + with ppn+acetate or with pan alone were harvested, washed and resuspended in 0.5 m sodium carbonate buffer, ph 9 at a cell density of 20-25 mg protein ml −1 . routine activity check was performed by measuring ammonia formation over time at ph 10 and 0.6 m total na + . activity and time course measurements with the determination of nitrile consumption and amide and acid production were carried out in 2-ml eppendorf tubes shaken on a thermotwister comfort shaker (quan-tifoil instruments) at a cell density of 0.1-0.6 mg protein ml −1 in either 20 mm citrate buffer ph 5.5, 0.1 m phosphate buffer ph 7 or 20 mm carbonate buffer ph 8.4 and at 5 or 25°c. reactions were initiated by the addition of nitriles (10 mm) in the presence of 4 mm internal standard (veratrol). the final reaction volume was 1.0 ml. regular samples were taken at regular time intervals and quenched in hcl-acidified mobile phase solution. after centrifugation of the denatured protein, the supernatant was injected directly on the high performance liquid chromatograph (hplc). samples for straight-phase hplc measurements were extracted with ethyl acetate and then dried over na 2 so 4 before injection. for the fed-batch experiments, 150 μl of halomonas sp. cell suspension (0.87 mg) was washed and resuspended in 0.1 m phosphate buffer ph 7 to a final volume of 1 ml in a 1.5-ml eppendorf tube. also, 12 μl of pure pan (89 mm) or 16 μl (100 mm) rac-phenylglycinonitrile was added to start the reaction. additional substrate was added only after full conversion was achieved. the reaction was carried out at 25°c and samples were taken periodically. in order to test the stability of the whole cell biocatalyst, resting cells were suspended in sodium carbonate buffer of ph 10 (20-25 mg protein ml −1 ) and subsequently stored on ice. during time the activity of the cells was determined by incubating a sample of the cell suspension in 20 mm citrate buffer of ph 5.5 with 10 mm pan and 2.5 mm of internal standard (veratrol) at 25°c. cell-free extract was obtained either by using an iks lab equipment constant cell disruption system at 2 kbar at room temperature or by osmotic shock after lysozyme treatment (1 mg ml −1 ). the cell debris and unbroken cells were removed by centrifugation at 30,000 ×g for 5 min. nitrile, amide, and carboxylic acid concentrations during the reaction were determined by hplc. the conversions of mandelonitrile, ppn, benzonitrile, and pan were followed on an alliance waters 2695 separation module equipped with a waters 2487 dual l uv absorbance detector using a 4.6×50 mm merck chromolith speedrod rp-18e reversed-phase column and h 2 o:acn (90:10)+0.1% (v/v) trifluoroacetic acid (tfa) as eluent (1 ml min −1 ). wavelength of the detector was set at 215 nm. the enantiomeric purity of the hydrolysis products of rac-mandelonitrile and rac-ppn was determined on an hplc system equipped with a waters 515 pump and a waters 486 tunable absorbance detector, which was set at 215 nm. separation was achieved using a straight-phase 4.6 × 250 mm chiralpak ad-h column with hexane: isopropanol 80:20 (rac-mandelonitrile) and 90:10 (rac-ppn)+0.1% (v/v) tfa as eluents (0.5 ml min −1 ). the conversion of rac-phenylglycinonitrile and the enantiopurities of the corresponding acids were determined using the same hplc module as above. separation was obtained using a daicel crownpak cr(+) column and h 2 o + 0.1% (v/v) hclo 4 as eluent (0.5 ml min −1 ). conversions of acrylonitrile and 3-cyanopyridine were followed on an hplc system with a waters 590 programmable hplc pump and a shimadzu spd-10a vp uv-vis detector. separation was achieved using three 4.6×50 mm merck chromolith™ speedrod rp-18e columns in series for acrylonitrile and a single column for 3-cyanopyridine. water+0.1% (v/v) tfa (acrylonitrile) or acetic acid (3-cyanopyridine) was used as eluent. the uv detector was set at 210 nm. ammonium and cell protein concentrations were detected spectrophotometrically according to weatherburn (1967) and lowry et al. (1951) . the isolation of the dna and subsequent determination of the g+c content were performed by the thermal denaturation/reassociation technique (marmur 1961; marmur and doty 1962) . genomic dna for phylogenetic analysis was extracted from the cells using the ultra-clean soil dna extraction kit (molbio laboratories, usa) following the manufacturer's instructions. the 16s rrna genes were amplified using general bacterial primers. the pcr products were purified from lowmelting agarose using the wizard pcr-prep kit (promega, usa) according to the manufacturer's instructions. sequencing was performed using the big dye terminator v.3.1 sequencing reaction kit on an abi 3730 dna automatic sequencer (applied biosystems, inc., usa). the sequences were first compared with those stored in genbank using the blast algorithm. the sequences were aligned with those from the genbank using clustalw. phylogenetic trees were constructed with four different algorithms using the treeconw program package (van de peer and de wachter 1994). no growth or ammonia formation was observed upon incubation of soda soil and soda lake sediments at ph 10 with ppn as the only substrate. however, when it was used as a nitrogen source in the presence of acetate as carbon and energy source, a positive enrichment culture was obtained from soda soils. the culture was dominated by fat nonmotile rods filled with refractive inclusions (supplementary fig. 1) . a pure culture, designated strain anl-αch3, was isolated from a single colony and proved to be able to utilize ppn as nitrogen source. phylogenetic analysis (fig. 1) placed the isolate into the genus halomonas in the gammaproteobacteria with haloalkaliphilic species h. campisalis and h. campaniensis as the closest relatives (97% sequence similarity). the genus halomonas accommodates a wide range of species characterized by high halo-and alkali-tolerance but the ability to utilize nitriles has never been demonstrated in this physiologically versatile group. growth of strain anl-αch3 with arylaliphatic nitriles at ph 10 the bacterium was not able to grow with ppn alone. when the latter was supplied at concentrations 0.5-4 mm in presence of 20 mm acetate, the growth occurred with the maximum specific growth rate of 0.12 h −1 , usually after a 20-30-h lag phase. ammonium was not detected in the culture supernatant. in contrast, pan supported the growth without an additional carbon and energy source and the final growth yield was proportional to the initial pan concentration up to 5 mm. higher pan concentrations inhibited growth of the bacterium. during the growth, pan was rapidly consumed releasing nearly stoichiometric amounts of ammonia during the early logarithmic growth phase with only a limited intermediate accumulation of phenylacetic acid (paa) (fig. 2) . the biomass production continued after complete pan disappearance, signifying the formation of an essential intermediate. hplc analysis, however, did not show any formation of phenylacetamide. high growth yield (biomass on 2 mm pan was equivalent to the yield on 10 mm acetate) indicated complete utilization of pan. the latter indicates an ability to utilize the aromatic ring, which is not an ordinary property of the genus halomonas. the capacity of the bacterium to metabolize the aromatic ring was confirmed in growth experiments with benzoic and salicylic acids. the culture, pre-grown on pan, started to grow with benzoate (up to 5 mm) and salicylate (up to 3 mm) after a 1-day lag phase. the growth resulted in the accumulation of a brownish soluble compound in both cases, which is in contrast to the pan culture ( supplementary fig. 2) . the same effect was observed in sterile control alkaline medium when it was incubated with catechol. this suggests that the oxidation of benzoic and salicylic acids was going through a diquinol with partial spontaneous side oxidation of the latter at highly alkaline conditions. in contrast, utilization of pan either did not result in substantial accumulation of autooxidizable intermediate(s) or proceeded through a different ring hydroxylation pathway. phenol (1-2 mm) halomonas anticariensis fp35 t , ay489405 halomonas maura s-31 t , aj271864 halomonas eurihalina atcc 49336 t , x87218 halomonas salina f8-11 t , aj295145 halomonas cupida dsm 4740 t , l42615 halomonas alimentaria ykj-16 t , af211860 halomonas pantelleriensis aap t , x93493 halomonas muralis lmg 20969 t , aj320530 did not support growth of the novel bacterium, in contrast to h. campisalis. one of the main purposes of this work was to investigate a possibility of bio-conversion of arylaliphatic nitriles at extremely haloalkaline conditions. the ph profile for growth with ppn as the nitrogen source indicated a moderate alkaliphilic nature of the novel isolate (fig. 3) . on the other hand, resting washed cells of strain anl-αch3, pre-grown at ph 10 with ppn and acetate, were able to actively hydrolyze nitriles within a much broader ph range from ph 5 and at least up to ph 11 with an optimum within the alkaline region (fig. 3) . resting cell incubation tests with 2-phenylpropionamide, phenylacetamide, and rac-mandelamide did not result in acid formation even after 20 h of incubation. as was mentioned before, intermediate amide formation was also not observed during growth of the organism on pan and ppn. these observations indicate that anl-αch 3 degrades aromatic nitriles through the nitrilase pathway. nine different aliphatic and aromatic nitriles with straight and branched side chains were tested for conversion by using washed cells of strain anl-αch 3 pre-grown with ppn. the influence of different αsubstitutes (and enantiomers) on the reaction rate was also investigated ( table 1) . as expected, the highest conversion rate was obtained with pan, since it was utilized as carbon source. the nitrilase proved to be strongly (r)-selective. the experimental results with (r)and (s)-mandelonitrile showed that the (r)-enantiomer is converted 20 times faster than the (s)-one (fig. 4a) . the strong but not absolute (r)-selectivity of anl-αch3 was confirmed in kinetic resolution tests with other chiral nitriles, such as phenylglycinonitrile and 2-phenylpropionitrile ( fig. 4b and c ). an interesting example of application of the nitrilase from anl-αch3 strain is in situ dynamic kinetic resolution of (r, s)-mandelonitrile (fig. 4d) . in the process, the unwanted enantiomer was racemized via the nitrile decomposition and chemical hydrocyanation pathway according to the following scheme: the fast racemization assured a constant supply of the desired (r)-enantiomer of cyanohydrin, which was then fully converted by the nitrilase into (r)-mandelic (+95% ee) acid in 2 h. a fed-batch experiment with pan demonstrated that the halomonas cells could convert 0.8 m of the nitrile into crystalline paa. the substrate was added in nine equal portions. every subsequent portion reached full conversion slower than the previous one (first portion was converted in <3.5 h and the eighth portion was fully converted in~27 h). the cells eventually stopped working after converting a part of the last, ninth, portion. in a similar experiment with phenylglycinonitrile, at least 0.2 m was converted into the corresponding rac-phenylglycine (fig. 4a ) and the cells would have probably converted more since there were no signs of inhibition by the final product. a 3-month storage test on ice demonstrated that the cells, when resuspended in sodium carbonate buffer of ph 10, are highly stable. no change in activity was observed during this time of storage. during cell disruption experiments, the cell-free preparations obtained by using osmotic shock resulted in better activity retention compared to disruption using rapid decompression. moreover, 85% of the activity was recovered in the extract when using anaerobic conditions, and 31% when using aerobic conditions during the osmotic shock. in contrast, rapid decompression resulted in only 8% activity recovery. this is the first report in literature of a haloalkaliphilic bacterium capable of utilizing arylaliphatic nitriles. the bacterium can hydrolyze several arylaliphatic nitriles at a ph as high as 10-10.5 and salt concentrations of up to 3.5 m total na + . most interesting is the capacity of the novel halomonas isolate to grow exclusively with pan. in fact, the potential to use phenol and its derivative carbonic acids at haloalkaline conditions has so far been shown only for a few halomonas strains and only one of them is haloalkaliphilic: the immediate relative of strain anl-αch3 h. campisalis (alva and peyton 2003) . but, in contrast to the latter, our isolate did not utilize phenol (1-5 mm). the exact pathway of the pan and ppn degradation in the novel strain needs special investigation. the ability of the isolate to grow with benzoic and salicylic acids suggests a potential for the complete aromatic ring degradation. absence of paa accumulation in the culture supernatant during growth on pan and a certain disbalance between pan hydrolysis and paa accumulation by washed cells indicated rapid utilization of the acid. however, while it is easy to explain for the growing culture, the reason for paa deficiency in washed cell experiments is not clear. our first hypothesis was that the washed cells could have rapidly oxidized the paa formed from pan. however, special anoxic test showed the same disbalance, while anoxic incubation with paa demonstrated no consumption. perhaps, the cells might contain some of the paa formed during intracellular pan hydrolysis. while pan is clearly a xenobiotic, paa is a natural compound with plant hormone activity which occurrence is related to phenylalanine metabolism. many bacteria can utilize this compound by activation with coa followed by ring hydroxylation (navarro-llorens et al. 2008 ). in any case, the exact mechanism of paa utilization in alkaliphilic halomonas strain needs further detailed analysis. assuming its r-enantioselectivity, the nitrilase from anl-αch3 might be useful in the (dynamic) kinetic resolution of phenylglycinonitrile (see fig. 4 ). the hydrolysis product, (r)-phenylglycine, is an intermediate in the synthesis of β-lactam antibiotics (mueller and huebner 2003) . the differences in anaerobic and aerobic cell disruption experiments revealed a negative influence of oxygen on the enzyme. the activity loss, caused by exposure to oxygen, might be caused by oxidation of the catalytic cysteine present in the anl-αch 3 nitrilase. this catalytic cysteine is present in all known nitrilases (mateo et al. 2006) . the stability of the free enzyme is an important issue. the nitrilase is unstable outside of the cell environment. unlike the whole cells, cell-free extracts from both disruption experiments quickly (after several days) lost most of their initial activity. the activity loss is not caused by proteases and it can be partially improved by adding glycerol to the storage medium. in conclusion, the data presented indicate the possibility of biodegradation and utilization of a broad range of arylaliphatic nitriles at a ph range between 5.5 and 11 by a novel haloalkaliphilic isolate, strain anl-αch3, presumably through nitrilase system. assuming unique phenotypic properties and distinct phylogeny, strain anl-αch3 is proposed as a new species halomonas nitrilicus sp. nov. fig. 4 biocatalytic properties of washed cells of strain anl-αch3. a hydrolysis of rac-phenylglycinonitrile with halomonas sp. resting cells at ph 8.4 and 25°c (diamonds, 2phenylglycinonitrile; squares, phenylglycine; circles, ee (r)phenylglycine). b hydrolysis of rac-mandelonitrile at ph 5.5 and 5°c (triangles, benzaldehyde; diamonds, mandelic acid; squares, mandelonitrile; circles, ee (r)-mandelic acid). c hydrolysis of rac-2phenylpropionitrile at ph 7 and 25°c (diamonds, 2phenylpropionitrile; squares, 2phenylpropionic acid; circles, ee (r)-phenylpropionic acid). d one pot chemo-enzymatic dynamic kinetic resolution synthesis of (r)-mandelic acid at ph 7 (squares, rac-mandelonitrile; diamonds, mandelic acid; triangles, benzaldehyde; circles, ee (r)-mandelic acid) description of halomonas nitrilicus sp. nov. (ni.tri'li.cus n.l. n. nitrilum nitrile, nitrile group; n.l. adj. nitrilicus nitrile-utilizing) nonmotile, gram-negative rods, 0.5×1.5-3 µm. utilizes phenylpropionitrile as nitrogen source and phenylacetonitrile as carbon, energy, and nitrogen source. able to hydrolyze a range of arylaliphatic and aliphatic nitriles using the nitrilase system. also utilizes as carbon and energy source the following substrates: acetate, propionate, butyrate, pyruvate, fumarate, succinate, citrate, malate, lactate, ethanol, glucose, maltose, sucrose, cellobiose, galactose, raffinose, trehalose, glycerol, mannitol, sorbitol, xylose, and glycogen. degrades and utilizes as sole growth substrates benzoate and salicylate. cannot grow anaerobically with nitrate, nitrite, or n 2 o. moderately alkaliphilic with a ph range for growth between 7.5 and 10.5 with an optimum at ph 9 and extremely salt tolerant, growing at salt concentrations from 0.1 to 3.5 m total na + with an optimum at 0.3-0.5 m. optimum growth temperature is 32°c. the g + c content in the genomic dna is 62.5 mol% (tm). the type strain is anl-αch3 t (nccb 100121 t =uniqem u241 t ) is isolated from soda soils in south-eastern siberia. the nearest cultured relative is halomonas campisalis. the genbank accession number of the 16s-rrna gene sequence of the type strain is eu447162. phenol and catechol biodegradation by the haloalkaliphile halomonas campisalis: influence of ph and salinity the nitrile-degrading enzymes: current status and future prospects industrial methods for the production of optically active intermediates biotransformation of nitriles by rhodococci recent developments in the stereoselective synthesis of α-aminoacids enzymatic routes to enantiomerically pure aromatic alpha-hydroxy carboxylic acids: a further example for the diversity of biocatalysis identification of isobutyronitrile and isobutyraldoxime o-methyl ether as volatile microbial catabolites of valine degradation of acetonitrile through a sequence of microbial reactors metalloenzyme nitrile hydratase: structure, regulation and application to biotechnology nitrile hydrolases enrichment strategies for nitrile-hydrolysing bacteria protein measurement with folin phenol reagent a procedure for isolation of dna from microorganisms determination of the base composition of deoxyribonucleic acid from microorganisms stabilisation of oxygen-labile nitrilases via co-aggregation with poly (ethyleneimine) economic aspects of amino acids production genetic analysis of phenylacetic acid catabolism in arthrobacter oxydans cect386 über das vitamin b 12 -bedürfnis phototropher schwefelbakterien d-phenylglycine hydrochloride acetonitrile degradation under haloalkaline conditions by natronocella acetinitrilica gen microbial isobutyronitrile utilization at haloalkaline conditions treecon for windows: a software package for the construction and drawing of evolutionary trees for the microsoft windows environment plant cyanogenic glycosides phenol-hypochlorite reaction for determination of ammonia acknowledgements this work was supported by nwo-rfbr (47.011.2004.010), nwo under cerc3, rfbr (07-04-00153), and by the program on molecular and cell biology ras.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012056-8b3xffsh authors: maas, ronald h. w.; bakker, robert r.; jansen, mickel l. a.; visser, diana; de jong, ed; eggink, gerrit; weusthuis, ruud a. title: lactic acid production from lime-treated wheat straw by bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate date: 2008-04-01 journal: appl microbiol biotechnol doi: 10.1007/s00253-008-1361-1 sha: doc_id: 12056 cord_uid: 8b3xffsh conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. in this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and bacillus coagulans dsm 2314. decrease in ph because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. after 55 h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for 55%, 75%, and 80%, respectively. lactic acid (40.7 g/l) indicated a fermentation efficiency of 81% and a chiral l(+)-lactic acid purity of 97.2%. in total, 711 g lactic acid was produced out of 2,706 g lime-treated straw, representing 43% of the overall theoretical maximum yield. approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a ca(oh)(2) suspension. of the lime added during the pretreatment of straw, 61% was used for the neutralization of lactic acid. this is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and ph control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime. lactic acid is used throughout the world in manufacturing of food, chemicals, and pharmaceutical products. recently, there is a lot of interest in biodegradable poly-lactic acid, which is an alternative to petrochemically derived plastic (drumright et al. 2000) . chiral pure lactic acid is produced commercially by microbial fermentation of the carbohydrates glucose, sucrose, lactose, and starch/maltose derived from feedstocks such as beet sugar, molasses, whey, and barley malt (narayanan et al. 2004 ). the choice of feedstock depends on its price, availability, and on the respective costs of lactic acid recovery and purification (datta et al. 1995; vaidya et al. 2005) . as an alternative to these traditional feedstocks, lignocellulosic biomass is an inexpensive and widely available renewable carbon source that has no competing food value. lignocellulose consists primarily of cellulose and hemicellulose; polymers build up of mainly hexose sugars and pentose sugars, which are embedded in a matrix of the phenolic polymer lignin. the main pathway to derive fermentable sugars from lignocellulose is through enzymatic hydrolysis by cellulolytic and hemicellulolytic enzymes. a mechanical and chemical pretreatment of the lignocellulose is required to reduce particle size, to modify and/or to remove the lignin, and with that to enhance the accessibility of the polysaccharides for enzymatic hydrolysis (claassen et al. 1999) . various chemical pretreatments of biomass have been studied in research and development of lignocellulose-to-ethanol production technology (mosier et al. 2005) . one is the use of lime (calcium hydroxide) at relatively mild temperature conditions (chang et al. 1998 ). lime as a pretreatment agent has promising potential because it is inexpensive, safe, and its use hardly results in sugar degradation products such as furfural and hydroxymethyl furfural. nevertheless, this alkaline pretreatment features a relatively high ph value (>10) of the treated biomass, and at these ph levels, the activity of common cellulolytic and xylanolytic enzymes, necessary for the depolymerization of (hemi)-cellulose, is negligible low. therefore, lowering the ph is essential to achieve an efficient enzymatic hydrolysis of the polysaccharides. one approach to remove calcium hydroxide is by washing the lime-treated biomass before enzymatic hydrolysis (chang et al. 1998 ); however, this leads to the use of high amounts of water. another way to lower the ph of the pretreated material is by neutralizing calcium hydroxide with sulfuric acid. yet, this results in the formation of the low value byproduct gypsum. as an alternative improvement to these approaches, we propose to use the calcium hydroxide present in limetreated biomass as neutralizing agent for organic acids produced in microbial fermentation processes. to examine this proposed concept, lime-treated wheat straw (ltws) was added fed-batch-wise during a simultaneous saccharification and fermentation (ssf) process in a 20-l controlled stirred fermenter containing hydrolytic enzymes and bacillus coagulans dsm 2314, a thermophilic bacterium capable to convert both hexoses and pentoses homofermentative to l(+)-lactic acid (otto 2004; patel et al. 2006) . the objective of this research was to evaluate whether high alkaline-treated lignocellulosic biomass (without neutralization) can be used directly in a ssf process by (1) providing a carbon source for enzymatic hydrolysis and fermentation and (2) providing a source of alkali to control the ph in the fermentation process. wheat straw was selected as a lignocellulose model feedstock and was purchased from a farm in the northeast of the netherlands. the wheat straw was air dried (89.5% [w/w] dry matter [dm] ) and ground through a 2-mm screen. the lime pretreatment was performed by filling two 15-l mixers (terlet, the netherlands), both with 1,650 g ground wheat straw, 13 kg tap water, and 165 g calcium hydroxide. this wheat straw suspension was heated and kept at 85°c for 16 h under continuously stirring at 30 rpm. the ltws suspension was subsequently cooled to 30°c, dehydrated by placing the ltws in a cotton bag, and pressing the suspension using a manual piston press at pressure up to 9.7 kg/m 2 . after dehydration, an amount of 11.45 kg ltws with an average dm content of 27.0% (w/w) and ph 11.8 was obtained and served as substrate for further experiments. the chemical composition of ltws was determined as described by van den oever et al. (2003) . the enzyme preparation gc 220 (genencor-danisco, rochester, usa) containing cellulase, cellobiase, and xylanase activity of 116, 215, and 677 u/ml, respectively (kabel et al. 2006) , was used for this study. the preparation had a specific gravity of 1.2 g/ml and contained 4.5 mg/ml glucose, 2.9 mg/ml mannose, and 0.8 mg/ml galactose. the bacterium b. coagulans strain dsm 2314 was used as the lactic acid-producing micro-organism. bacterial cells were maintained in a 10% (w/w) glycerol stock solution and stored at −80°c. chemicals, unless indicated otherwise, were purchased from merck (darmstadt, germany). gelrite plates were prepared with a medium containing (per liter): glucose, 10 g; gelrite, 20 g (duchefa, haarlem, the netherlands); yeast extract, 10 g (duchefa); (nh 4 ) 2 hpo 4 , 2 g; (nh 4 ) 2 so 4 , 3.5 g; bis-tris, 10 g (usb, ohio, usa); mgcl 2 6h 2 o, 0.02 g; and cacl 2 .2h 2 o, 0.1 g. glucose and gelrite were dissolved in stock solution a (four times concentrated). the ph of this stock solution was adjusted to 6.4 with 2 m hydrochloric acid and autoclaved for 15 min at 125°c. the remaining nutrients were dissolved in stock solution b (1.33 times concentrated), which was also adjusted to ph 6.4 with 2 m hydrochloric acid but was filter sterilized (cellulose acetate filter with pore size of 0.2 μm, minisart, sartorius). after sterilization, the medium was prepared by combining stock solutions a and b and gelrite plates were poured. the bacteria were cultivated on gelrite plates for 48 h at 50°c. an isolated colony was used to inoculate a 100-ml broth with similar composition and preparation as described above but without the addition of gelrite. the culture was incubated statically for 24 h at 50°c and functioned as the inoculum for a 1,400-ml broth. this culture was incubated also statically for 12 h at 50°c and served as a 10% (v/v) preculture for the ssf experiments. the ssf of ltws was carried out in a 20-l fermenter (applikon, schiedam, the netherlands) with ph and temperature control (biocontroller adi 1020). at the start of ssf, the fermenter was filled with 6.0 kg tap water and 1,400 g dehydrated ltws (dm content of 27.0% [w/w]). the following nutrients were then added to the ltws suspension: yeast extract, 150 g (duchefa); (nh 4 ) 2 hpo 4 , 30 g; (nh 4 ) 2 so 4 , 52.5 g; mgcl 2 6h 2 o, 0.3 g; and cacl 2 2h 2 o, 1.5 g. the ltws suspension was then heated to 50°c, and the ph was adjusted to 6.0 with 101 g 3 m sulfuric acid (~30 g h 2 so 4 ). the ssf process of ltws to lactic acid consisted of three phases: (1) the prehydrolysis phase of preloaded ltws, (2) the fed-batch phase with automatic feeding of ltws from a screw feeder, and (3) the batch phase with ph control by a calcium hydroxide suspension and no ltws feeding. a schematic representation of the experimental setup is shown in fig. 1 . the prehydrolysis was initiated by the addition of 40 ml enzyme preparation (88 mg enzyme/g dm substrate) to the ltws suspension and was incubated for 2 h at 50°c under continuously stirring at 250 rpm. the fed-batch phase was initiated by the addition of 1,500-ml preculture of b. coagulans dsm 2314 to the fermenter. the lactic acid produced by the bacteria was neutralized by the automatic addition of 8,623 g dehydrated ltws (dm of 27.0%) to the fermenter through a feeder (k-tron soder feeders, canada) and was regulated by the ph of the medium, which was set at 6.0. throughout the fed-batch phase, an amount of 280 ml of enzyme preparation (total enzyme loading of 98 mg/g dm substrate) was added proportional to the ltws addition rate into the fermenter. during the batch phase, the ph was controlled at 6.0 by the addition of 20.0% (w/v) calcium hydroxide suspension. samples were withdrawn for dm, substrate, and (by)product analysis. for the analysis of monomeric sugars, the fermentation broth samples were centrifuged (3 min at 17,400×g), and the ph of the supernatant was adjusted to 5.0 with barium carbonate using a ph indicator (bromophenolblue) followed by filtration of the liquid. the analysis was performed by high-performance anion-exchange chromatography using a carbopack pa1 column (column temperature of 30°c) and a pulsed amperometric detector (ed50; dionex, sunnyvale, ca, usa). before injection, the system was equilibrated with 25.5 mm naoh for 10 min at a flow rate of 1.0 ml/min. for the separation of monomeric sugars, at injection, the mobile phase was shifted to deionized water for 30 min. postcolumn addition of sodium hydroxide was used for detection of the neutral monomeric sugars. the determination of soluble oligomeric sugars was performed by centrifugation for 5 min at 3,000 rpm (centaur 2, beun de ronde, the netherlands) of preweighed samples and freeze drying the supernatant overnight. pellets were weighed and hydrolyzed with sulfuric acid, and neutral monomeric sugars were determined according to the method as described by van den oever et al. (2003) . for the calculations, an average molecular weight of oligomers from glucan and xylan of 166 and 132 g/mol, respectively, were applied, resulting in a hydrolysis factor of 1.08 and 1.14, respectively. for the analysis of insoluble polymeric sugars, samples of 25 g were centrifuged for 5 min at 3,000 rpm (centaur 2, beun de ronde); the supernatant was removed, and the pellet was washed by resuspension in 25 ml fresh demineralized water followed by a centrifugation step of 5 min at 3,000 rpm (centaur 2, beun de ronde). the sequence of resuspension and centrifugation was repeated three times. after the last removal of the supernatant, the pellets were freeze dried overnight. the pellets were weighed (values used for dm calculation), polymeric material was hydrolyzed with sulfuric acid, and neutral sugars were analyzed according to the method as described by van den oever et al. (2003) . for the calculations, a molecular weight of glucan and xylan of 162 and 132 g/mol, respectively, were applied, resulting in a hydrolysis factor of polymer to monomer of 1.11 and 1.14, respectively. the analysis of organic acids was performed by highpressure liquid chromatography according to the procedure described by maas et al. (2006) . the chiral purity (%) of lactic acid was determined by derivatization of all lactates using methanol, after which both enantiomers of methyl lactate were separated on a chiral gas chromatography column and detected using a flame ionization detector. the chiral purity was expressed as the area of the main enantiomer divided by the sum of areas of both enantiomers. the theoretical maximum lactic acid (la theor. max . [g]) production was calculated according the following equation (eq. 1): where dm substrate =the total dry matter of substrate ltws (g), f polysacch. =fraction polysaccharides per substrate (g/g), hf monsacch./polysacch. =hydrolysis factor of polysaccharides, incorporation of water results in 1.11 g hexose from 1.00 g glucan and 1.14 g pentose from xylan and arabinan (g/g), and ff=fermentation factor of 1.00 g lactic acid per gram of monomeric sugar. the efficiency of the enzymatic hydrolysis (%, w/w) was based on the amount of hydrolyzed polysaccharides (g; calculated by the difference between initial amounts and analyzed insoluble amounts) divided by the amount of polysaccharides (g) initially present in the substrate. the fermentation efficiency (%, w/w) is expressed as the amount of lactic acid produced (g) divided by the amount of monomeric sugars consumed (g) by the bacteria. the overall efficiency of the ssf (%, w/w) was calculated by the amount of lactic acid produced (g) divided by the theoretical maximum amount of lactic acid (g) determined as described in eq. 1. the polysaccharide composition of the ltws consisted mainly of glucan, xylan, and arabinan of 33.0%, 19.0%, and 2.0% (w/w), respectively, whereas the remaining mass constituted of lignin, ash, extractives, and uronic acids. some of the soluble components in wheat straw were partially removed by the solid/liquid separation (dehydration) of the ltws. the focus of this study was on the conversion of glucan, xylan, and arabinan, which are the predominant polysaccharides present in ltws and accounted for 99.8% (w/w) of the total polymeric sugars. previous work showed that the cellulase preparation gc 220, used for the saccharification of polysaccharides, functioned optimally at 50°c and ph 5.0 (maas et al., submitted for publication), whereas growth conditions for b. coagulans dsm 2314 were 54°c and ph 6.5 (otto 2004) . in this study, both the enzymatic hydrolysis and the fermentation occurred simultaneously in the same reactor at compromising conditions, which were set at 50°c and ph 6.0. the ssf of ltws to lactic acid was studied in a 20l controlled stirred fermenter. previous results showed that when this process was performed without a prehydrolysis of an initial amount of ltws, the concentration of monomeric sugars was low and resulted, therefore, in relatively low lactic acid productivity. as a consequence, the fed-batch addition rate of the alkaline substrate to neutralize the produced lactic acid was low (results not shown). to start the fermentation with a substantial initial amount of fermentable sugars (>2 g/l), a prehydrolysis of 378 g ltws and enzyme preparation (88 mg per g dm ltws) in approximately 6 l volume at ph 6.0 for 2 h was introduced. this resulted in glucose, xylose, and arabinose concentrations of 2.0, 0.4, and 0.3 g/l, respectively (fig. 3a) . the second phase (ii) was initiated by introducing a 1,500-ml preculture of b. coagulans dsm 2314. a minor amount of lactic acid produced in the preculture caused a slight ph decrease and was automatically neutralized by the addition of ltws (fig. 2a,b) . after a lag phase of 4 h, the dissolved oxygen concentration decreased rapidly within 1 h from 100% to oxygen-limiting conditions of below 1% (results not shown), and lactic acid production started. at that moment, concentrations of glucose, xylose, and arabinose of 3.3, 0.7, and 0.3 g/l, respectively, were present (fig. 3a) . these sugars were consumed simultaneously where glucose was utilized faster than xylose and arabinose. simultaneous with the consumption of these monomeric sugars, lactic acid was produced, which was neutralized by the automatic addition of alkaline ltws. by the addition of alkaline substrate throughout the fedbatch phase, the ph was maintained accurately at 6.0±0.1 (fig. 2a,b) . at the end of phase ii, a total amount of 10,023 g dehydrated ltws (~2,706 g dm ltws) and 320 ml of enzyme preparation was added to the fermenter. a lactic acid concentration of 20.5 g/l supernatant was detected (fig. 3b ), corresponding to a total of 342 g lactic acid. the chiral l(+) purity of lactic acid was determined at 99.4%, which is similar to that obtained with xylose as the sole carbon source (otto 2004) . at the end of phase i, a low acetic acid concentration was detected in the medium, which increased to 1.5 g/l throughout phase ii but remained constant during phase iii (results not shown). this indicates that acetic acid was most likely not a fermentation product formed by b. coagulans. acetic acid can be released upon solubilization and hydrolysis of hemicellulose during chemical pretreatment (palmqvist et al. 1999) . by the dehydration procedure of the ltws, part of the acetic acid was easily separated from the substrate by removing the press water. apparently, a remaining amount of acetic acid was fed together with the substrate to the fermenter. furthermore, minor traces of other organic acids such as succinic acid and formic acid (<0.5 g/l) were detected in the fermentation broth. phase iii was initiated by changing the ph control from the addition of alkaline ltws to a 20% (w/v) calcium hydroxide suspension. to maintain the ph at 6.0, the addition of calcium hydroxide suspension occurred relatively fast but shifted, however, after a few hours to a lower addition rate indicating a decline of the volumetric lactic acid productivity (figs. 2b, 3b) . to exclude limitation (e.g., by inactivation) of enzymes, an extra dosage of enzyme preparation (80 ml) was added to the fermenter after 23.5 h of incubation. this resulted immediately in a slight acceleration of the calcium hydroxide addition rate indicat-ing an increased lactic acid productivity and limitation of enzymatic activity (fig. 3b) . nevertheless, after 29.7 h of incubation, a decline of the calcium hydroxide addition rate was observed again. therefore, a second extra dosage of the enzyme preparation (240 ml) was added and resulted this time in a slight accumulation of glucose and xylose of 1.5 and 1.0 g/l (fig. 3a) , respectively, indicating that microbial conversion instead of enzymatic hydrolysis was rate limiting. after 32 h of incubation, a lactic acid concentration of 37.1 g/l was obtained, with a chiral l(+)lactic acid purity of 99.4%. continuation of the ssf process to a total incubation period of 55 h resulted in a slightly increased lactic acid concentration of 40.7 g/l supernatant (~37.8 g lactic acid/kg fermentation broth) with an overall volumetric lactic acid productivity of 0.74 g l −1 h −1 . at this stage, a chiral l(+)-lactic acid purity of 97.2% was analyzed. this slight decline in lactic acid purity is possibly a result of infection with other undesired lactic acidproducing microorganisms. because the substrate used fig. 2 control of ph (a) during simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation gc 220 and b. coagulans dsm 2314 (b). the areas between the dotted lines represent the prehydrolysis phase (i), the fedbatch phase (ii) with ph control by addition of alkaline ltws and enzymes, and the batch phase (iii) with ph control by addition of ca (oh) 2 suspension. extra enzyme preparation gc220 was added at the times indicated by the arrows was not sterile and also the chemical pretreatment and fermentation occurred in an open system under nonsterile conditions, microbial contamination throughout the ssf process is possible. the efficiency of the enzymatic hydrolysis of the polymeric material present in ltws is shown in fig. 4 . the insoluble polymeric fraction was determined at various time points throughout the ssf experiment. at the end of the prehydrolysis (2 h) of 378 g ltws, 36% of the insoluble glucan (fig. 4a) , 55% of xylan (fig. 4b) , and 62% of arabinan (fig. 4c) were converted to soluble saccharides including monomeric sugars and oligomeric sugars. after the fed-batch phase (13 h), 2,706 g ltws was added and resulted in a conversion of 42% of glucan, 57% of xylan, and 63% of arabinan to products including soluble saccharides and lactic acid. between 13 and 32 h of incubation, further hydrolysis of the polymeric sugars was observed. however, during the last 23 h of the ssf, minor hydrolysis of the polysaccharides occurred, and this corresponded with the decline in lactic acid productivity during this phase. after 55 h, 398 g of glucan, 130 g of xylan, and 11 g of arabinan was still present as insoluble polymeric material. with these values, the hydrolysis efficiency of the initial glucan, xylan, and arabinan present in ltws were calculated as 55%, 75%, and 80%, respectively. the monomeric sugars, derived from the ltws, were partly converted to lactic acid (711 g) by b. coagulans and accounted for 81% (w/w) of the theoretical maximum, indicating the formation of other products such as microbial biomass and carbon dioxide. an overall conversion yield of 43% (w/w) of the theoretical maximum was calculated according to eq. 1. the fate of polysaccharides initially present in ltws after 55 h of incubation is shown in table 1 . a part of the polysaccharides present in ltws remained as insoluble polysaccharides (37% w/w), whereas a minor part was converted into soluble oligomeric (5% w/w) and monomeric (3% w/w) sugars. another part of the initial polysaccharides present in the ltws was not recovered in the form of saccharides or lactic acid and was therefore ascribed as 'unaccounted.' the lactic acid produced (342 g) during the fed-batch phase (ii) was neutralized with alkaline-pretreated wheat straw. presented values are averages based on duplicate analytical measurements. a total of glucan, xylan, and arabinan b part of the initial polysaccharides remained present as insoluble polysaccharides. c part of the initial polysaccharides was not recovered and therefore denoted as 'unaccounted.' during this phase, an amount of 2,328 g ltws was added to the fermenter. together with this substrate, an amount of 230 g calcium hydroxide was added to the fermenter and accounted for a ratio of 0.67 g calcium hydroxide per gram of lactic acid. the lactic acid (369 g) produced during the batch phase (iii) was neutralized with 163 g calcium hydroxide resulting in a ratio of 0.44 g lactic acid per gram calcium hydroxide. lignocellulosic feedstocks are considered as potential attractive substrates for the production of bulk chemicals. pretreatment of biomass is required to break open the lignocellulosic matrix, and an enzymatic hydrolysis is necessary for the hydrolysis of polymeric carbohydrates. the lime pretreatment has proven to enhance enzymatic digestibility of the polysaccharides present in lignocelluloses (chang et al. 1998; kaar and holtzapple 2000) and results, in comparison to other pretreatment routes, in minor inhibitor formation. however, before the enzymatic hydrolysis, it is essential to adjust the ph to a level optimal for enzymatic activity. in this study, the reduction in ph by washing or neutralization was omitted by using the alkaline character of ltws to neutralize lactic acid produced by microbial fermentation during a ssf process. the results showed that the largest part of the polysaccharides in ltws was converted enzymatically and the resulting sugars were fermented simultaneously to mainly lactic acid by b. coagulans dsm 2314. between 10 and 30 h of incubation, the bacteria utilized the monomeric sugars, as soon as they appeared in the medium, resulting in relatively low monomeric sugar concentrations (<2 g/l). this indicates that throughout this period, the enzymatic hydrolysis was the rate-controlling step. the highest lactic acid productivity was observed during the fed-batch phase and the initial hours of the batch phase and declined rapidly after approximately 20 h of incubation, as shown in fig. 3b . an extra addition of enzyme preparation showed a slight improvement of the volumetric lactic acid productivity but shifted within a few hours again to a relatively low production rate. a second extra enzyme addition did not affect the lactic acid productivity significantly (fig. 3b ). this addition of new enzymes resulted in a modest liberation of hemicellulose sugars (xylose, arabinose), but no further hydrolysis of glucan occurred. this shows that the remaining glucan was too recalcitrant or not accessible for further hydrolysis, resulting in decreasing lactic acid productivity. another possible explanation of the decreased lactic acid productivity is the inhibition of enzymes and/or bacteria by the increasing lactic acid concentration. a lactic acid concentration of 40.7 g/l supernatant (~37.8 g lactic acid/kg fermentation broth) with a relatively high chiral purity was determined after 55 h of incubation, corresponding to an overall lactic acid yield of 43% of the theoretical maximum. moreover, the efficiencies of the enzymatic saccharification and the fermentation were both determined. these calculations showed that, based on residue analysis, at the end of the ssf process (55 h), 55% of the glucan, 75% of the xylan, and 80% of the arabinan present in ltws was enzymatically hydrolyzed, which agree well with previously obtained results from experiments aiming to convert ltws to ethanol. to improve the yield, it is necessary to decrease the recalcitrance or improve the accessibility of polymeric sugars in the ltws by optimization of the pretreatment procedure. the concentrations of soluble monosaccharides and oligosaccharides in the medium were relatively low, which can be expected in a ssf process. a fermentation yield of 81% was determined (related to the amount of monosaccharides released from the ltws) and is slightly better than the results obtained by otto (2004) who reported the production of 35 g/l lactic acid from 50 g/l xylose as the sole carbon source. because no other soluble fermentation products were detected, the remaining 19% of the ltwsderived monomeric sugars were most presumably converted to bacterial biomass and some carbon dioxide during the aerobic part of the fermentation. several process parameters can be listed for enhancement of the overall lactic acid yield and productivity such as improving the accessibility of polysaccharides by a more severe lime pretreatment, enzyme dosage, type of enzymes, b. coagulans strain, size and growth phase of inoculum, ph gradient in ssf, and in situ product removal of lactic acid. these issues will be subject to further studies. during the fed-batch phase (ii), it was possible to counterbalance the ph decrease caused by lactic acid production by the addition of the alkaline feedstock. this suggests that it is possible to combine lime treatment with the production of other organic acids from lignocellulosic biomass. throughout this phase, the ratio of calcium hydroxide in ltws added per produced lactic acid was determined at 0.67 g/g. the theoretical stoichiometric neutralization of 1.00 g lactic acid requires 0.41 g calcium hydroxide. therefore, only 61% of the calcium hydroxide initially added to the wheat straw was used for lactic acid neutralization. on the other hand, throughout the batch phase (iii), an alkaline/acid ratio of 0.44 g/g was calculated corresponding to 93% of the added calcium hydroxide suspension used for lactic acid neutralization. the low efficiency of the calcium hydroxide added with the ltws for lactic acid neutralization during phase ii has three possible explanations. first, part of the calcium hydroxide could have been used during the chemical pretreatment of the wheat straw such as the neutralization of acetic acid or other organic acids and/or irreversible binding to the lignin. second, the calcium hydroxide might be released slowly from the insoluble wheat straw fibers and could therefore partly have been used for lactic acid neutralization in the fed-batch phase. finally, besides lactic acid production, other acidification reactions could have contributed to the decrease in ph and therefore the demand of alkaline substrate, for instance, the decrease in ph caused by the consumption of ammonium as the nitrogen source by microorganisms (guebel et al. 1992) . the results in this paper show that it is possible to use lignocellulosic materials for the production of lactic acid. lignocellulosic biomass is a relatively inexpensive substrate, and this affects feedstock costs for lactic acid production positively. nevertheless, in comparison to the traditional relatively 'clean' feedstocks with a well-defined composition, using heterogenic lignocellulosic substrates will require a more intensified downstream processing (dsp) to recover and purify the lactic acid from the complex fermentation broth. the costs of feedstock materials and operational costs of the dsp contribute considerably to the overall production costs of lactic acid (åkerberg and zacchi 2000) . whether the cost decrease in using lignocellulosic feedstocks outweighs the potential increasing costs of dsp was not analyzed at the moment. in summary, ltws was converted into l(+)-lactic acid by b. coagulans throughout a ssf process at a 20-l bench scale. the pentose and hexose sugars derived from the polymeric material were utilized simultaneously by b. coagulans resulting in a final lactic acid concentration of 40.7 g/l supernatant, which accounted for 43% (w/w) of the theoretical yield. to our knowledge, this is the first paper demonstrating a process having a combined alkaline pretreatment of lignocellulosic biomass and ph control in organic acid fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime. an economic evaluation of the fermentative production of lactic acid from wheat flour lime pretreatment of crop residues bagasse and wheat straw utilisation of biomass for the supply of energy carriers technological and economic potential of poly(lactic acid) and lactic acid derivatives polylactic acid technology influence of the nitrogen source on growth and ethanol production by pichia stipitis nrrl y-7124 using lime pretreatment to facilitate the enzymic hydrolysis of corn stover standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials lactic acid production from xylose by the fungus rhizopus oryzae features of promising technologies for pretreatment of lignocellulosic biomass l(+) lactic acid fermentation and its product polymerization preparation of lactic acid from a pentose-containing substrate. patent wo hahn-hägerdal b (1999) main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeast isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid switchgrass (panicum virgatum l.) as a reinforcing fibre in polypropylene composites acknowledgments this project is supported with a grant of the dutch programme eet (economy, ecology, technology), a joint initiative of the ministries of economic affairs, education, culture, and sciences and of housing, spatial planning and the environment. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012072-98wuq5ri authors: sun, yvonne; gustavson, ruth l.; ali, nadia; weber, karrie a.; westphal, lacey l.; coates, john d. title: behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors date: 2009-06-17 journal: appl microbiol biotechnol doi: 10.1007/s00253-009-2051-3 sha: doc_id: 12072 cord_uid: 98wuq5ri the response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. the observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. we observed attraction toward nitrate when dechloromonas aromatica strain rcb and azospira suillum strain ps were grown with nitrate. when d. aromatica and dechloromonas agitata strain ckb were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. when a. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. a gene replacement mutant in the perchlorate reductase subunit (pcra) of d. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. washed-cell suspension studies revealed that the perchlorate grown cells of d. aromatica reduced both perchlorate and nitrate, while a. suillum cells reduced perchlorate only. based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by d. aromatica. to the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. it clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly. electronic supplementary material: the online version of this article (doi:10.1007/s00253-009-2051-3) contains supplementary material, which is available to authorized users. perchlorate (clo 4 − ), a predominantly synthetic compound manufactured in the form of ammonium salt, is used primarily as an energetics booster or oxidant in solid rocket fuels and munitions (motzer 2001) . however, it has been known to inhibit mammalian thyroid hormone production by blocking iodide uptake (stanbury and wyngaarden 1952) . recently, it was demonstrated that perchlorate was translocated through the na+/i− symporter, which can be found in thyroid and lactating breast tissues and subsequently caused iodine deficiency and accumulation of perchlorate in milk (dohan et al. 2007 ). long-term exposure to perchlorate has been implicated in thyroid hormone deficiency, a known cause of impaired neuropsychological fetal and infant development (porterfield 1994; howdeshell 2002) . because of historical legal discharge of unregulated manufacturing waste streams, disposal pond leachate, and the periodic servicing of military inventories, the widespread presence of perchlorate in the environment poses a significant health threat. perchlorate is unique in its chemical stability and high solubility. therefore, remediation efforts for perchlorate contamination have primarily focused on either in situ or ex situ biological treatment technologies (urbansky 1998; coates and achenbach 2004; coates and jackson 2008) based on the ability of some bacteria to reductively respire perchlorate completely to innocuous chloride in the absence of oxygen. many dissimilatory perchlorate-reducing bacteria (dprb) are now in pure culture with the dominant species in mesophilic environments belonging to the dechloromonas and azospira genera of the betaproteobacteria (coates and achenbach 2004) . all known dprb are non-fastidious and exhibit a broad range of metabolic capabilities. they are facultative anaerobes or microaerophiles, and most of them alternatively respire nitrate (coates and achenbach 2004) . although dprb are found to be ubiquitous , little is known of their abilities to access perchlorate in their natural environment. three pure cultures from the environmentally dominant dechloromonas and azospira genera were chosen in this study. dechloromonas aromatica strain rcb and dechloromonas agitata strain ckb were previously isolated from aquatic sediment ) and paper mill waste, respectively, , while azospira suillum strain ps was isolated from swine waste lagoon sludge . all three organisms are nonfermentative, motile, facultative anaerobes that reduce chlorate and perchlorate [(per)chlorate] coupled to the oxidation of simple organic acids and alcohols at circumneutral ph. these organisms are unable to utilize hydrogen as electron donor or fe(iii) and sulfate as alternative electron acceptors achenbach et al. 2001; coates et al. 2001) . in contrast to d. aromatica and a. suillum, d. agitata does not grow by dissimilatory nitrate reduction chaudhuri et al. 2002) . as a continuation of our ongoing studies into this unique form of metabolism, we investigated the behavioral response of these three organisms to structurally analogous electron acceptors and identify their ability to distinguish between nitrate, chlorate, and perchlorate. all cultures were grown on phosphate-buffered (ph 7) basal freshwater medium containing (per liter): nh 4 cl (0.25 g), k 2 hpo 4 (1.07 g), kh 2 po 4 (0.52 g), a vitamin stock solution (10 ml), and a mineral stock solution (10 ml). the vitamin stock solution contained the following (per liter): biotin (2 mg), folic acid (2 mg), pyridoxine hcl (10 mg), riboflavin (5 mg), thiamine (5 mg), nicotinic acid (5 mg), pantothenic acid (5 mg), vitamin b12 (0.1 mg), p-aminobenzoic acid (5 mg), and thioctic acid (5 mg). the mineral stock solution contained (per liter) the following: nitrilotriacetic acid disodium salt (1.5 g), mgso 4 .7h 2 o (3.0 g), mnso 4 .h 2 o (0.5 g), nacl (1.0 g), feso 4 .7h 2 o (0.1 g), cacl 2 .2h 2 o (0.1 g), cocl 2 .6h 2 o (0.1 g), zncl (0.13 g), cuso 4 .5h 2 o (0.01 g), alk(so 4 ) 2 .12h 2 o (0.01 g), h 3 bo 2 (0.01 g), na 2 moo 4 .2h 2 o (0.025 g), nicl 2 .6h 2 o (0.024 g), na 2 wo 4 .2h 2 o (0.025 g), and na 2 seo 4 (0.02 g). acetate was the sole electron donor, and one of oxygen, nitrate (10 mm), chlorate (10 mm), or perchlorate (10 mm) was used as the sole electron acceptor as required. d. aromatica was grown at 30°c, while d. agitata and a. suillum were grown at 37°c, the respective temperature optima for these organisms. microbial perchlorate reduction is accomplished with the action of several enzymes, including perchlorate reductase (pcr) encoded by the pcrabcd operon and chlorite dismutase (cld) encoded by a single open reading frame cld. a deletion mutation in the pcra gene was made as previously described (bender et al. 2005) . briefly, a 357-bp region upstream of pcra start codon was amplified by polymerase chain reaction (pcr) and cloned in between saci and spei sites in pbluescript ii ks+ (stratagene), creating ppcra1. a 323-bp region downstream of pcra stop codon was amplified by pcr and cloned in between ecori and xhoi in ppr1, resulting in ppcra2. finally, the knockout construct ppcra3 was generated by cloning the 1.6-kb tetracycline resistance cassette from pbbr322 into spei and ecori sites in ppr2. the deletion of pcra gene in the knockout mutant was confirmed by pcr. the resultant mutant that is incapable of growth or reduction of perchlorate or chlorate (bender et al. 2005) was used to determine the role of perchlorate reductase in taxis towards (per)chlorate and nitrate. each of the pcra mutant and wild-type d. aromatica culture was grown with limited amounts of oxygen as the electron acceptor in phosphate-buffered (10 mm, ph 7) basal freshwater media in sealed serum bottles supplemented with 20 mm acetate as the electron donor and 2 mm perchlorate to induce the perchlorate reduction pathway. the limiting amount of oxygen was supplied as 10 ml of air injected through a sterile 0.22-μm nylon membrane filter into a sealed serum bottle (100 ml) containing 50 ml of liquid culture. inoculated bottles were horizontally agitated overnight at 30°c to maximize oxygen diffusion. induction of the perchlorate reduction pathway was confirmed by reverse transcription pcr (rt-pcr) determination of the presence of transcript for chlorite dismutase (cld) in the pcra mutant grown with perchlorate relative to a control grown in media without perchlorate ( supplementary fig. 1 ). cells were collected by filtration onto a 0.2-μm filter. total rna was prepared directly from the filters using the trizol reagent (invitrogen, number 15596) following manufacturer's protocol. for each sample, 0.2 μg of total rna extract was used for complementary dna (cdna) synthesis of pcra and cld transcripts using reverse primers (5′-cgccgatgtatctcttcatgttcac-3′ for pcra and 5′-tgaatggttccgagcgttgtcggac-3′ for cld) and the moloney murine leukemia virus reverse transcriptase (promega, number m1701) following manufac-turer′s protocol. then, 1 μl of cdna synthesis product was used as template in 20 cycles of pcr amplifications for each gene of interest. parallel reactions lacking the reverse transcriptase were set up for each sample as controls for amplification from genomic dna contamination. a standard swarm plate assay (adler 1966 ) was adapted to establish the ability of all three organisms to navigate in a gradient of the electron donor, sodium acetate. plastic petri dishes containing 25 ml of phosphate buffered (ph 7) basal freshwater medium amended with 1 mm sodium acetate and 0.3% noble agar were prepared. cells of d. aromatica, d. agitata, and a. suillum were inoculated in the middle of the agar plates and incubated aerobically at 30°c for d. aromatica and at 37°c for d. agitata and a. suillum. the size of swarm ring was recorded 48 h after inoculation. agar plate-based assay cells were grown to mid-log phase and harvested by centrifugation at 6,000 rpm for 10 min in centrifuge bottles that were flushed with nitrogen gas. the cell pellet was washed and resuspended in fresh anoxic basal media without any electron donor or acceptor. all liquid transfers were performed on the bench top under a stream of nitrogen gas to avoid any contact with atmospheric oxygen. the prepared cell suspensions were transferred using n 2 gas-flushed syringes into 25 ml anaerobic phosphate-buffered basal media with 1% noble agar in the molten state to give a final cell count of approximately 1×10 10 cells·ml −1 . the inoculated molten agar bottles were amended with either an electron donor (10 mm) or acceptor (10 mm) as required and an appropriate concentration of chloramphenicol to inhibit de novo protein synthesis (shaw 1983 ) and growth during the assay incubation. growth studies with each organism amended with a range of chloramphenicol concentrations indicated that the optimum working concentrations for d. aromatica, d. agitata, and a. suillum were 2.0, 1.0, and 2.5 mg·l −1 , respectively (data not shown). at these concentrations, cell motility, as observed by phasecontrast microscopy, remained unaffected, indicating the presence of active metabolism to support flagellar rota-tions. in contrast, growth of these cells, as measured by optical density (data not shown), was completely inhibited. the chloramphenicol-amended cell suspensions were poured into sterile petri dishes in an anaerobic glove bag under a n 2 -h 2 (95:5, vol/vol) atmosphere. once solidified, sterile filter paper discs (whatman number 2, 6 mm in diameter) saturated with a sterile anaerobic aqueous stock (1 m) of the chemical of interest were placed on the surface of the agar, allowing the development of a concentration gradient through diffusion of the chemical into the agar. the prepared plates were incubated at room temperature anaerobically inside the glove bag or aerobically on the bench top. a response was identified by the formation of a tan/white cell-dense halo ( supplementary fig. 2 ) around the discs after 2 h or overnight incubation for the electron donor and electron acceptor assays respectively caused by the attraction of the motile cells toward the chemical. the presence of a visible halo around the filter disc was scored as a positive result (denoted by + in table 1 ), while the absence of a halo in the same time frame was scored as a negative result (denoted by − in table 1 ). a capillary-based assay using the palleroni chamber (palleroni 1976) was also adapted to provide quantitative measurement of the behavioral response. anaerobic cell suspensions were prepared as described above and added to the palleroni chamber including the channel and both wells. a pre-cut capillary (3-cm length, 1.46-mm outer diameter, 1.12-mm inner diameter) was filled with chemical of interest at a final concentration of 10 mm and carefully placed in the channel connecting both wells allowing cells to enter the capillaries from both open ends. each chemical was tested in two sets of triplicates such that one set of capillaries was immediately removed from the chamber to establish a baseline, while a second set of capillaries was removed 15 min later. once removed from the chamber, each capillary was rinsed carefully to remove cells adhered to the exterior of the tube and purged of its content into a sterile microcentrifuge tube. the amount of cell mass in the microcentrifuge tube was estimated by measuring protein content with a microbicinchoninic acid assay kit (pierce, number 23235), which allows for measurement of protein concentration in the micromolar range. a positive response was identified by an increase in the measured protein content inside the capillary after 15 min of incubation relative to the protein content in the capillary tubes with brief exposure to the cells. cultures of d. aromatica and a. suillum grown with acetate (10 mm) and perchlorate (10 mm) were anaerobically harvested at late log phase by centrifugation. cell pellets were washed with anaerobic phosphate buffer (10 mm, ph 7) twice and resuspended in the same phosphate buffer. the suspensions were added to anaerobic phosphate buffer amended with 10 mm acetate and 10 mm of electron acceptor (nitrate, chlorate, or perchlorate) and incubated at 30°c and 37°c for d. aromatica and a. suillum, respectively. samples were taken out at different time points, and concentrations of anions were analyzed. concentrations of perchlorate, chlorate, chloride, nitrate, and nitrite were analyzed by ion chromatography as previously described (chaudhuri et al. 2002) . the recently finished genome of d. aromatica (nc_ 007298) (http://genome.jgi-psf.org/finished_microbes/ decar/decar.download.html) revealed the genetic potential for a motility behavioral response in perchlorate-reducing bacteria. based on the annotated genome, d. aromatica contains three sets of gene clusters for flagella biosynthesis suggesting flagella-based motility. three sets of gene clusters located on chromosome (bp# 790489-810487, 1244490-1254769, and 4194360-4191281) were annotated to encode chemotaxis signal transduction proteins, including multiple copies of chea, cheb, ched, and cher. in addition, 22 putative chemotaxis sensory transducer genes were apparent, five of which were located in the three chemotaxis gene clusters. the remaining chemotaxis sensory transducer genes were dispersed throughout the genome occasionally in gene clusters unrelated to motility or chemotaxis. phase-contrast microscopy revealed that all three organisms were motile when grown under planktonic conditions either aerobically or anaerobically with nitrate or (per)chlorate as electron acceptors. to initially investigate the ability of these organisms to respond to a chemical gradient, a traditional swarm plate assay was utilized with acetate (1 mm) as the limiting growth substrate in the agar under aerobic conditions. swarm rings were visible around the inoculation site 24 h after inoculation. within 48 h, migration of swarm rings had advanced to a diameter of 1.02±0.08 cm for d. aromatica, 0.94±0.05 cm for d. agitata, and 2.68±0.33 cm for a. suillum, indicating a motility response in these organisms to a substrate concentration gradient. in the agar plate-based assay, appropriate concentrations of chloramphenicol were amended to ensure the observed celldense halo was a result of bacterial accumulation from migration in the agar instead of growth. different concentrations of perchlorate were tested with a. suillum grown anaerobically on acetate as the sole electron donor and perchlorate as the electron acceptor to determine assay efficiency. the size of the halo was dependent on the concentration of perchlorate used in the filter discs with a lower limit of approximately 50 mm for a detectable response ( supplementary fig. 3) . as a result, all subsequent plate-based assays were performed with 1 m stock solution of the chemical of interest onto the filter discs for the most pronounced observable result. initial studies indicated that inclusion of an electron donor such as acetate in the agar plate was a prerequisite for a motility response toward an electron acceptor, suggesting that active cell metabolism was required. no taxis response was observed with sulfate ( supplementary fig. 2) , which is not an electron acceptor utilized by these organisms achenbach et al. 2001; coates et al. 2001) . no taxis response toward any of the anaerobic electron acceptors (nitrate, perchlorate, chlorate) was observed if agar plates were prepared anaerobically but incubated aerobically on the bench top. however, each of the dprb tested in this study was attracted toward the electron donor acetate under aerobic incubation regardless of the prior growth conditions (data not shown). from the agar plate-based assay, both d. aromatica and a. suillum, grown under nitrate-reducing conditions, showed acetate-dependent motility toward nitrate but not (per)chlorate (table 1) . when grown with perchlorate, both organisms were attracted toward both chlorate and perchlorate, while perchlorate-grown d. aromatica was also attracted to nitrate (table 1) . surprisingly, while d. agitata is incapable of growth by nitrate reduction , this organism also responded readily toward nitrate when grown with perchlorate (table 1 ). in contrast to both the dechloromonas species, perchlorate-grown a. suillum did not respond to nitrate (table 1) demonstrating the unique ability of a. suillum to distinguish between structurally analogous nitrate and (per)chlorate when establishing a physiological response. when acetate in the agar was replaced by an electron acceptor and was added instead onto the filter discs, similar behavioral patterns were observed: perchlorate-grown a. suillum responded toward acetate only when (per)chlorate was amended in the agar, while perchlorate-grown d. aromatica responded toward acetate when nitrate or (per)chlorate was amended in the agar. identical results were observed when each organism was grown under chlorate-reducing conditions. in the capillary-based assay using the palleroni chamber (palleroni 1976) , 10 mm acetate was amended in the chamber and the capillary tube in order to observe a behavioral response. acetate-dependent responses were observed using 10 mm of electron acceptor in the capillary tube after 15 min of incubation. as such, no chloramphenicol was amended because of the short incubation time, which is lower than the minimum doubling time of approximately 90 min observed for any of these organisms (chaudhuri et al. 2002) . acetate-only samples were included as a negative control to account for random migration of cells into the capillary tube. the capillarybased assay using the palleroni chamber yielded similar results as the agar plate-based assay. in the case of d. aromatica grown with perchlorate, a fivefold increase in protein content inside the capillary tube filled with either (per)chlorate or nitrate was apparent compared to time zero and acetate-only samples (fig. 1a) . this confirms that perchlorate grown d. aromatica cells were unable to distinguish these anions and were attracted to each of the three electron acceptors (fig. 1a) . in contrast, when a. suillum was grown under identical conditions with perchlorate as the sole electron acceptor, the protein content within the capillary tube filled with nitrate did not increase relative to the control. however, increases in protein content were observed if the capillary tube was filled with (per)chlorate (fourfold and sixfold for perchlorate and chlorate, respectively) further demonstrating the ability of this organism to distinguish these analogous compounds (fig. 1b) . similar to agar plate-based assay, perchlorate-grown d. agitata also responded to nitrate and (per)chlorate even though the organism does not grow by dissimilatory nitrate reduction nitrate (fig. 1c) . as part of a previous study on the genetics of perchlorate reductase, a d. aromatica mutant in which the gene coding for the alpha subunit of perchlorate reductase, pcra, was replaced by a kanamycin-resistance cassette (bender et al. 2005) . the pcra mutant lacks a functional perchlorate reductase and thus was unable to grow anaerobically with (per)chlorate but was still able to grow aerobically and anaerobically with nitrate (bender et al. 2005) . to determine if perchlorate reductase is necessary for taxis toward (per)chlorate, motility behavior between wildtype and pcra mutant was compared by growing both strains overnight with a limited supply of oxygen and 2 mm perchlorate for inducing expression of the perchlorate reduction pathway after the depletion of oxygen. mobility of both strains grown under these conditions was confirmed with phasecontrast microscopy and induction of the (per)chlorate reduction pathway was confirmed by rt-pcr of the chlorite dismutase gene (cld) (supplementary fig. 1 ). using the agar plate-based assay where acetate was amended in the agar, wild-type d. aromatica was attracted toward nitrate and (per)chlorate, while pcra mutant was only attracted toward nitrate but not (per)chlorate (table 1) , suggesting that an active perchlorate reductase is necessary for taxis to (per)chlorate. (per)chlorate and nitrate reduction by active cell suspensions washed cell suspension experiments were performed to test the inherent ability of d. aromatica and a. suillum to reduce nitrate and (per)chlorate under non-growth conditions. when grown with perchlorate, suspensions of d. aromatica readily reduced approximately 4.2 mm perchlorate to 1.1 mm chlorate and 3.3 mm chloride (fig. 2a) . additionally, these same cell suspensions completely reduced 10 mm nitrate to equimolar nitrite within 4 h (fig. 2b) , supporting the inability of this organism to distinguish between perchlorate and nitrate when grown with perchlorate. in contrast, when grown with nitrate, suspensions of d. aromatica did distinguish between the alternative electron acceptors and only reduced nitrate but not (per)chlorate (fig. 2c) . unlike d. aromatica, suspensions of perchlorate-grown a. suillum did not reduce nitrate but readily reduced (per)chlorate (fig. 2d) , again demonstrating the ability of this organism to discriminate between these two compounds regardless of growth conditions. these studies provide evidence that dprb and microorganisms of any type can sense and show a behavioral response to a concentration gradient of chlorine oxyanions in their environment. furthermore, these results also demonstrate that the response is species-specific. bacterial motility response toward oxygen as an electron acceptor has been studied extensively in multiple organisms, and transducers such as aer have also been identified (taylor et al. 1999 ). however, the phrase "electron acceptor taxis" was first coined for attraction toward nitrate and fumarate in escherichia coli and salmonella typhimurium after anaerobic induction of the corresponding reduction pathway and the repelling effects from some respiratory inhibitors and uncouplers (taylor et al. 1979 ). subsequently, more organisms were reported to be attracted toward anaerobic electron acceptors. for example, rhodobacter sphaeroides 2.4.3 showed a nitrite reductase-dependent attraction toward nitrate and nitrite (lee et al. 2002) . shewanella oneidensis strain mr-1 was also attracted toward many diverse electron acceptors out fig. 1 capillary assay in palleroni chamber using a dechloromonas aromatica, b azospira suillum, and c dechloromonas agitata grown anaerobically with acetate (10 mm) and perchlorate (10 mm). the results depicted are the average of triplicate samples of more than a dozen that the organism is capable of utilizing for growth (nealson et al. 1995; bencharit and ward 2005) . it was established in r. sphaeroides that motility response to light, oxygen, and the alternative electron acceptor dimethyl sulfoxide (dmso) was dependent on the electron transport. it was proposed that an electron transport intermediate potentially served as the response signal instead of an actual chemical attractant (armitage et al. 1985; gauden and armitage 1995) . such responses were categorized as "energy taxis" because the electron transport chain is part of a cellular energygenerating system. in energy taxis, bacteria sense the stimuli through the internal redox state reflected by changes in the electron transport chain and transduce the signal through the common chemotaxis signaling pathway (taylor and zhulin 1998; alexandre and zhulin 2001) . alexandre et al. conclusively demonstrated electron transport as the stimulus for energy taxis in the diazotrophic azospirillum brasilense (alexandre et al. 2000) and described criteria to identify this type of response (alexandre and zhulin 2001) . first, chemicals interacting directly with the electron transport should elicit a response. second, there should be a correlation between the magnitude of the response with the physiology of electron transport chain. third, mutations that surrender activity in a terminal reductase for a particular substrate will disrupt the electron flow leading to the substrate, subsequently resulting in a loss of the attraction response. a brief survey in the genome of d. aromatica revealed strong potential for this organism to display taxis behavior such as the presence of flagellar biosynthesis genes and multiple copies of genes known to be involved in chemotaxis signal transduction. in this study, several lines of evidence led us to propose energy taxis as the basis for the observed responses by d. aromatica. first, the requirement of an electron donor such as acetate for any behavioral response to electron acceptors suggests a metabolism-dependent behavior where reduction of electron acceptors is necessary to elicit a response. exchanging the placement of acetate and electron acceptors tested between the agar and the filter discs in the agar plate-based assay yielded the same behavioral pattern, suggesting the involvement of the electron transport such that both the input of reducing equivalents and the output of electrons fig. 2 reduction of perchlorate and nitrate in washed cell suspensions of (a, b, c) dechloromonas aromatica and (d) azospira suillum grown anaerobically with acetate (10 mm) as the electron donor and 10 mm perchlorate (a, b, d) or 10 mm nitrate (c) as the electron acceptor. the results depicted are the average of triplicate samples onto acceptors can directly or indirectly serve as response signals. the inhibition of oxygen on the response to alternative electron acceptors excludes the possibility of nitrate or (per)chlorate-specific chemoreceptors and further suggests the involvement of the electron transport chain in which oxygen was interrupting electron transport onto nitrate or (per)chlorate by inhibiting nitrate and perchlorate reductases, respectively (chaudhuri et al. 2002; coates and achenbach 2004) . second, chemicals that elicit positive motility response were also the physiological substrates for each dprb tested in washed cell suspensions under non-growth conditions. nitrate was the only electron acceptor to elicit a positive motility response in d. aromatica grown with nitrate and the only electron acceptor that was reduced by nitrate-grown d. aromatica in washed cell suspensions. similarly, both nitrate and (per)chlorate elicited a positive response in d. aromatica grown with perchlorate, and in washed cell suspensions, (per)chlorate was reduced to chloride and nitrate was readily reduced to nitrite. accumulation of nitrite in the suspension suggested the absence of an active nitrite reductase in perchlorate grown cells and also suggested that perchlorate reductase from d. aromatica was responsible for overlapping reduction of (per)chlorate and nitrate. a similar dual activity of the perchlorate reductase was previously observed in d. agitata, which cannot grow on nitrate (chaudhuri et al. 2002) and was further demonstrated in this study by the behavioral response of d. agitata to nitrate and (per)chlorate. in fact, perchlorate reductase purified from d. agitata is known to be capable of reducing both nitrate and (per) chlorate (coates and heinnickel, unpublished data) further supporting this conclusion. the same correlation between behavioral response and physiology (i.e., reduction of specific electron acceptors) was also evident in a. suillum. when grown with nitrate, a. suillum was attracted toward nitrate but not (per)chlorate and reduced only nitrate in washed cell suspensions. when grown with perchlorate, a. suillum was attracted toward (per)chlorate and reduced only (per)chlorate but not nitrate in washed cell suspensions. if perchlorate reductase was central in mediating both the motility response and reduction in cell suspensions, then a. suillum harbors a unique homolog of perchlorate reductase, which can successfully differentiate between nitrate and (per)chlorate and reduce (per)chlorate only. interestingly, a pcr primer set recently designed based on known pcra sequences to detect dprb in the environment failed to amplify pcra gene in a. suillum (nozawa-inoue et al. 2008) , supporting the distinctiveness of a. suillum perchlorate reductase apart from the known dechloromonas perchlorate reductases. moreover, the difference could be reflected in unique amino acid residues attributing to the loss of nitrate-reducing capability and failure of detection by the canonical primer set. nevertheless, the connection between behavioral response and physiological reduction capacity of each dprb tested in this study strongly supported energy taxis as the underlying mechanism in which reduction of nitrate and (per)chlorate gradients by corresponding terminal reductases coupled to oxidation of acetate created the signal to elicit the response. finally, to demonstrate the necessity of terminal reductases in the observed behavior, the response of a d. aromatica perchlorate reductase mutant to (per)chlorate was tested. unable to grow on (per)chlorate, the pcra mutant was grown with a limited amount of oxygen in the presence of (per)chlorate to induce the perchlorate reduction pathway. using agar plate-based assay, the mutant cells failed to show attraction to (per)chlorate while the wild-type cells under the same conditions were readily attracted to (per)chlorate, further supporting the requirement of an active perchlorate reductase to establish a motility response toward (per)chlorate in d. aromatica. interestingly, it was observed that the perchlorate reductase mutant was attracted to nitrate suggesting that the nitrate reductase was possibly induced when the culture growth conditions became anaerobic even though nitrate was not present. together, these results demonstrate that dprb are capable of metabolism-dependent movement toward (per)chlorate with energy taxis as the potential underlying mechanism in d. aromatica. these results also suggested for the first time a possible difference in substrate range for perchlorate reductases in different dprb. while d. aromatica and d. agitata have perchlorate reductases that reduce nitrate and (per)chlorate, a. suillum, however, has a unique homolog of perchlorate reductase that can potentially distinguish between these electron acceptors and reduce (per)chlorate but not nitrate. phylogenetic comparisons between perchlorate reductases with or without nitrate-reducing capability can provide important insight into key residues in (per)chlorate reduction as well as in the evolutionary history of perchlorate reductases in the dmso reductase family. moreover, the specificity of recognition may offer a unique opportunity to develop a (per)chlorate-specific biosensor using perchlorate reductase from a. suillum that avoids interferences from nitrate. ultimately, this study demonstrates the potential for dprb to seek out perchlorate in the environment during active perchlorate reduction, strengthening the support for using dprb in the treatment of perchlorate contamination. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. dechloromonas agitata gen. nov., sp. nov. and dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position chemotaxis in bacteria more than one way to sense chemicals energy taxis is the dominant behavior in azospirillum brasilense role of proton motive force in phototactic and aerotactic responses of rhodopseudomonas sphaeroides chemotactic responses to metals and anaerobic electron acceptors in shewanella oneidensis mr-1 identification, characterization, and classification of genes encoding perchlorate reductase reduction of (per) chlorate by a novel organism isolated from paper mill waste environmental factors that control microbial perchlorate reduction microbial perchlorate reduction: rocket-fuelled metabolism in situ bioremediation of perchlorate in groundwater the ubiquity and diversity of dissimilatory (per) chlorate-reducing bacteria anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of dechloromonas the na+/i symporter (nis) mediates electroneutral active transport of the environmental pollutant perchlorate electron transport-dependent taxis in rhodobacter sphaeroides a model of the development of the brain as a construct of the thyroid system taxis response of various denitrifying bacteria to nitrate and nitrite perchlorate: problems, detection, and solutions anaerobic electron acceptor chemotaxis in shewanella putrefaciens quantitative detection of perchloratereducing bacteria by real-time pcr targeting the perchlorate reductase gene chamber for bacterial chemotaxis experiments vulnerabilities of the developing brain to thyroid abnormalities: environmental insults to the thyroid system chloramphenicol acetyltransferase-enzymology and molecular biology effect of perchlorate on the human thyroid gland in search of higher energy: metabolismdependent behavior in bacteria electron acceptor taxis and blue light effect on bacterial chemotaxis aerotaxis and other energysensing behavior in bacteria perchlorate chemistry: implications for analysis and remediation acknowledgements support for this research to jdc was from grant daca72-00-c-0016 from the us department of defense serdp program. key: cord-276575-jfug80yu authors: aigner, achim title: applications of rna interference: current state and prospects for sirna-based strategies in vivo date: 2007-04-25 journal: appl microbiol biotechnol doi: 10.1007/s00253-007-0984-y sha: doc_id: 276575 cord_uid: jfug80yu within the recent years, rna interference (rnai) has become an almost-standard method for in vitro knockdown of any target gene of interest. now, one major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. from the mechanism, it becomes clear that small interfering rnas (sirnas) play a pivotal role in triggering rnai. thus, the efficient delivery of target gene-specific sirnas is one major challenge in the establishment of therapeutic rnai. numerous studies, based on different modes of administration and various sirna formulations and/or modifications, have already accumulated promising results. this applies to various animal models covering viral infections, cancer and multiple other diseases. continuing efforts will lead to the development of efficient and “double-specific” drugs, comprising of sirnas with high target gene specificity and of nanoparticles enhancing sirna delivery and target organ specificity. after antisense technologies and ribozymes, in the late 1990s a novel mechanism for gene-targeting was discovered: rna interference (rnai). it soon became clear that rnai represents a particularly efficient and-at least in vitro-easy-to-use method for the knockdown of the expression of a selected target gene. consequently, rnai is now a wellestablished method for high-throughput analyses as well as for functional studies in vitro, including mammalian cells. many pathological conditions rely on the aberrant expression of endogenous normal or mutant genes causing, e.g., alterations in signal transduction pathways, cellular proliferation, apoptosis, or resistance toward external factors. additionally, the infection of an organism can lead to the introduction and expression of foreign genes. while the inhibition of the activity of (aberrant) gene products, e.g., through small molecule inhibitors or inhibitory antibodies is one major focus in therapy, much attention has now shifted to an earlier step, i.e., the initial knockdown of the specific gene of interest through rnai. however, for the in vivo application of rnai in mammals as a therapeutic approach for reversing a pathological condition as well as for the study of particular gene functions, sophisticated strategies for the induction of rnai are needed. rnai is a naturally occurring, sequence-specific mechanism for gene silencing. its discovery in the nematode c. elegans (fire et al. 1998 ) was awarded the 2006 nobel prize for physiology or medicine. however, soon it became obvious that rnai, although somewhat more complicated, also exists in higher organisms including mammals. rnai relies on an intracellular multistep process, which can roughly be divided into the initiation phase (see below) and the subsequent effector phase. in the effector phase ( fig. 1, left) , which represents the actual rnai mechanism, small, 21-23 bp double stranded rna molecules (small interfering rnas, sirnas), are incorporated into the rna-induced silencing complex (risc; hammond et al. 2000) . upon adenosine triphosphate (atp)-dependent unwinding of the double-stranded sirna molecule through an rna helicase activity into a single-stranded, so-called guidance rna (nykanen et al. 2001) , the now activated risc (risc*) binds to its target mrna molecule (martinez et al. 2002; nykanen et al. 2001 ). this process is mediated through the sequence-specific hybridization of the guidance rna to the mrna target site and brings risc into close proximity to its target mrna molecule, which is then cleaved by the risc nuclease argonaute 2 (ago 2) and rapidly degraded due to its now unprotected ends (liu et al. 2004; rand et al. 2004; rivas et al. 2005 ). since risc is recovered for subsequent rounds, this represents a catalytical process leading to the selective reduction in specific mrna molecules and thus resulting in decreased expression of the targeted gene. the mechanism also demonstrates the pivotal role of sirna molecules in initiating rnai and established the delivery of sirna molecules as sufficient for rnai induction (elbashir et al. 2001a, b) . in a natural, experimental or therapeutical setting, sirnas can be directly or as precursor molecules introduced into a target cell through different strategies (fig. 1, upper part) . this includes viral or nonviral delivery of dnas, which are transcribed into long, double-stranded rna molecules. in the so-called initiation phase, these dsrnas are cleaved into sirnas by the multiprotein complex "dicer", which con-tains an n-terminal helicase domain, an rna-binding socalled piwi/argonaute/zwille (paz) domain, two rnase iii domains and a double-stranded rna binding domain (bernstein et al. 2001; collins and cheng 2005) . commercially available systems explore this mechanism by providing dna vector constructs coding for short hairpin rnas (shrnas): the double-stranded region of the shrna is formed through hairpin formation and intramolecular hybridization and is recognized by dicer, leading to the formation of sirnas homologous to the target gene of interest. alternatively, shrna molecules can be directly introduced into the cell. however, one major disadvantage of long double-stranded rna molecules, either directly introduced or intracellularly transcribed, is the induction of a cellular immune response through activation of the interferon system. the direct delivery of sirna molecules into the target cell strategy largely avoids this problem, although some interferon-stimulating sequences are known as well. furthermore, it does not require the action of dicer (bridge et al. 2003; hornung et al. 2005; sledz et al. 2003) . systematic studies on targeting efficacies have shown that optimal sirnas can be deduced according to certain selection rules. this includes an optimal length of 19-25 bp and a guanine-cytosine content between 30 and 52%, symmetric two nucleotide 3′ overhangs as well as specific nucleotides at certain positions (see, e.g., dykxhoorn and lieberman 2006 for review). based on these already established criteria, several computer-based algorithms allow the identification of optimal sirna sequences for any given gene of interest. one example is the sirna design software (sds) by the university of hong kong which combines algorithms from different companies and is accessible through the internet (http://i.cs.hku.hk/~sirna/ software/sirna.php). nevertheless, any presumably optimal sirna still requires extensive testing. this refers to a high targeting efficacy, which is, among others, also determined by variations in the accessibility of the target mrna at different positions, as well as to the absence of any unwanted side effects. in fact, nonspecific silencing of genes due to only partial sequence homology has been described (jackson et al. 2003) . furthermore, in vivo some sirna sequences, as well as longer dsrna molecules, have been shown to activate the innate immune system leading to nonspecific effects due to the stimulation of inflammatory responses (heil et al. 2004; judge et al. 2005; sioud 2005; sledz et al. 2003) . this phenomenon seems to depend on the presence of gu-rich sequences as well as on the formulation and amount of sirnas (heidel et al. 2004; ma et al. 2005; sioud and sorensen 2003) , and these aspects need to be considered for any therapeutic sirna application in vivo. since the discovery of the pivotal role of sirnas for inducing rnai (elbashir et al. 2001a, b) , the direct application of sirna molecules has been explored in vitro and in vivo. in vitro, several transfection reagents allow the delivery of sirnas in mammalian cells in the presence or absence of serum. the in vivo application of sirnas, however, requires the development of more sophisticated formulations and/or the identification of optimal modes of administration (see below). several proof-of-principle studies have shown the delivery of fluorophor-labeled sirna molecules into various organs (see, e.g., bradley et al. 2005a, b; pirollo et al. 2006; sioud and sorensen 2003) . beyond that, the specific in vivo knockdown of artificially introduced reporter genes like gfp or luciferase, or various endogenous target genes, has been described. the target organ was often the liver, but gene targeting in other organs, in other parts of the body, or in tumor xenografts has been reported as well (table 1) . taken together, these studies provide valuable insights into the delivery and efficacy of sirnas for the induction of rnai. beyond the detection of the downregulation of an endogenous target gene, the sirna-mediated rnai for therapeutical purposes has been explored. target organs include liver, kidney, lung, eye, ear, heart, pancreas, tumors, blood, as well as the central nervous system, and the peritoneum (see table 1 for an overview), using locally or systemically administered sirnas in various formulations. a large body of studies refers to the treatment of cancer with the primary goals being the inhibition of tumor growth. target molecules usually represent genes that have been shown previously to be relevant or rate-limiting for tumor growth, including growth factors and receptors as well as antiapoptotic or downstream signal transduction proteins in tumor cells. typically, these studies involve subcutaneous or orthotopic xenografts of different tumor entities in mice, and employ various strategies for local or systemic administration of a wide variety of sirna formulations (see below). in some cases, the antiangiogenic effect after sirna delivery to tumor endothelial cells, rather than an inhibitory effect on tumor cells, was explored (santel et al. 2006a, b) , or simultaneous targeting of tumor growth and tumor angiogenesis both contributed to the antitumorigenic effects observed (grzelinski et al. 2006 ). additionally, in some studies the blockage of cancer metastasis, e.g., to the lung, liver or bone has been achieved. taking into consideration that a large number of cancer patients die from metastases rather than the primary tumor, this represents another very relevant approach in cancer therapy. several studies focus on viral gene products, taking into consideration that options for protection or therapy through current antiviral drugs or vaccination strategies are rather limited. thus, novel rnai-based approaches to battle viral infections rely on the specific sirna-mediated knockdown of virus-specific genes. animal models infected with various viruses including hepatitis virus, influenza virus, respiratory syncytial virus (rsv), sars corona viruses, or ebola virus have been employed, and primary goals were the reduction in virus titers and protective effects including, when lethal doses were applied, prolonged survival rates upon specific sirna treatment. beyond studies related to cancer or viral infection, several target molecules with proven relevance in other pathologies have been selected. examples include fas in hepatitis, vascular endothelial growth factor (vegf) in macular degeneration due to extensive ocular neovascularization or tumor necrosis factor alpha (tnf-α) in arthritis, and these studies aim at the establishment of novel, improved therapeutic avenues through sirna-mediated gene silencing. as it can be seen from table 1 , some target genes are relevant in more than one pathology (e.g., fas in fulminant hepatitis, in renal ischemia-reperfusion injury or in hemorrhagic shock and sepsis in the lung; vegf in age-related macular degeneration or in tumor growth/tumor angiogenesis; caspase-8 in liver failure or in sepsis), and thus, specific sirnas may represent drugs applicable for the treatment of different diseases. some studies also compare sirna-based therapeutic strategies with already established drugs and discuss decreased side effects and/or higher specificity, or additive effects upon combination of both. other papers rather aim at the further elucidation of physiological processes through in vivo knockdown of a certain target gene. examples include oatc3 in blood-brain barrier transport, v2r in water and sodium homeostasis, or ho-1 in lung ischemia-reperfusion injury (table 1) . it should be noted that, although the therapeutic potential of sirnas has only been explored in the recent years, first sirna-based drugs are already in clinical trials. this includes aln-rsv01 (alnylam pharmaceuticals) for targeting the human rsv after viral infection, which is the first example of an antivirus sirna-based therapeutic in a phase i clinical study. other companies aiming at the development of rnai therapeutics for viral diseases include nastech/galenea, which are expected to start clinical trials in 2007. benitec, in collaboration with city of hope in duarte, california, has developed a multi-rna therapeutic for treatment of aids lymphoma. furthermore, age-related macular degeneration (amd) was treated with sirna-027 (sirna therapeutics) targeting the vegf receptor vegfr1 (shen et al. 2006) , and resulted in stabilization or even improvement of visual acuity (whelan 2005) . a 24month phase ii study to evaluate multiple doses of sirna-027 (also termed agn211745) in the treatment of subfoveal choroidal neovascularization associated with amd is currently recruiting patients. targeting the ligand (vegf) rather than its receptor, cand5 (acuity pharmaceuticals) has been employed for treatment of the same disease, and in the so-called c.a.r.e™ trial showed no adverse effects related to the drug. cand5, which is now named bevasiranib, was the first sirna to enter both phase i and ii clinical trials. recently, sr pharma plc announced the start of a phase i clinical trial with rtp-801i, an sirna therapeutic licensed from its subsidiary atugen ag that targets a gene product involved in the progression of amd. the same company has most recently announced that the fda has approved an investigational new drug (ind) application for a second sirna therapeutic, akii-5 being developed for the treatment of acute kidney injury. akii-5 is expected to reduce the frequency of postsurgery acute kidney injury in high-risk patients undergoing major cardiovascular surgery. finally, after successful completion of experiments demonstrating its therapeutic efficacy in animal models of pancreatic cancer, atu027 is scheduled to enter human clinical trials in 2007. in addition, several other pharmaceutical or biotechnology companies are pursuing collaborative or internal projects for the development of drugs based on rnai (see, e.g., behlke 2006 for review). advantages of the direct application of sirnas, rather than dna-based constructs coding for long dsrna, include the relatively easy chemical synthesis of small rna molecules, the lower probability of nonspecific side effects (see above) and the safety due to the fact that sirna delivery is based on nonviral transfer strategies and sirnas cannot integrate into the genome. on the other hand, successful sirnabased gene targeting relies on several preconditions: protection of the rather instable sirna molecules from nucleolytic degradation by serum nucleases, efficient cellular uptake and subsequent intracellular release into the cytoplasm, as well as the absence of intracellular immune responses, in vivo toxicity or rapid elimination in liver or kidney. strategies for the in vivo application of sirna molecules include local as well as systemic modes of administration as detailed in table 2 . however, many studies rely on the use of relatively high amounts of sirnas. bearing in mind that intracellular immune responses have been shown to be concentration-dependent, this may increase the risk of nonspecific effects in addition to other side effects and cost considerations. when sirnas are administered locally, lower doses are sufficient since nonspecific delivery to other organs as well as renal or hepatic elimination are reduced. this approach, however, is invasive and limited to tissues that are sufficiently accessible. with regard to systemic application, several studies rely on the hydrodynamic transfection of sirnas, i.e., the rapid (∼20 s) high-pressure injection of large volumes (up to 2 ml) of sirna-containing solution. hydrodynamic injection has led to the efficient induction of rnai in liver as well as in kidney, lung, pancreas, and spleen and is probably due to the transient enhancement of membrane-permeability. however, in animals, side effects have been observed (zhang et al. 2004a, b) , and in man, this method is not applicable at all. many groups have employed different approaches for the formulation of sirnas in carrier systems (table 3) , which deliver their sirna "payload" into the target tissue and target cell, some of them already being known as dna delivery techniques in gene therapy or antisense targeting. various liposomes/cationic lipids can be considered as examples of nonviral envelopes that protect sirnas, thus increasing serum stability, reducing renal excretion and mediating sirna uptake into the cells through endocytosis. the comparison of neutral versus cationic liposomes also reveals that the biodistribution as well as the uptake into macrophage seems to be dependent on their charge (landen et al. 2005; miller et al. 1998) , thus emphasizing the need for the further development and analysis of different liposomal particles. snalps (stable nucleic acid lipid particles) have been used for sirna-mediated targeting of an ebola-virus-specific gene (geisbert et al. 2006) or apob. this is also the first study that describes the systemic efficacy of formulated sirnas in a nonrodent species (zimmermann et al. 2006) . nanoparticles another strategy allowing the protection and cellular delivery of sirnas is the formation of nanoparticles with positively charged macromolecules. based on electrostatic interactions, complexes are formed with atelocollagen (banno et al. 2006; minakuchi et al. 2004; takei et al. 2004; takeshita et al. 2005) , chitosan (pille et al. 2006) , or polyethylenimine (pei). peis are synthetic linear or branched polymers available in a wide range of molecular weights (godbey et al. 1999; tang and szoka 1997) . due to the presence of a protonable amino group in every third position, leading to a high cationic charge density at physiological ph, peis are able to form noncovalent complexes with dna as well as small rna molecules like sirnas (urbanklein et al. 2005) or ribozymes (aigner et al. 2002) . this sirna complexation results in the complete protection against degradation in the presence of serum or rnase a and allows the efficient cellular uptake of the pei/sirna complexes through endocytosis. for any sirna formulation, the release from endosomes is critical for sirna delivery. in the case of pei, the so-called "proton-sponge effect" postulates improved transgene delivery by cationic complexes, which contain h + -buffering polyamines, based on enhanced endosomal cl − accumulation and subsequent osmotic swelling and lysis (behr 1997; boussif et al. 1995) . this effect may also apply for pei-mediated sirna delivery into the cytoplasm. additionally, to further enhance the efficacy of pei complexes through membrane-destabilization, the conjugation of melittin analogs to pei has been described (boeckle et al. 2005 (boeckle et al. , 2006 shir et al. 2006) . it should be noted, however, that by far not all peis are suitable for the transport of nucleic acids like sirnas (hassani et al. 2005; werth et al. 2006) . table 2 in vivo application of sirnas for the induction of rnai: modes of administration of naked or formulated sirnas hydrodynamic transfection bradley et al. 2005a, b; duxbury et al. 2004; giladi et al. 2003; hamar et al. 2004; heidel et al. 2004; hino et al. 2006; klein et al. 2003; lewis et al. 2002; liang et al. 2005; matsui et al. 2005; merl et al. 2005; sato et al. 2005; song et al. 2003; tompkins et al. 2004; zender et al. 2003 intravenous (without high pressure) bradley et al. 2005a, b; chien et al. 2005; ge et al. 2004; hassan et al. 2005; miyawaki-shimizu et al. 2005; morrissey et al. 2005a, b; schiffelers et al. 2004; soutschek et al. 2004; yano et al. 2004; takeshita et al. 2005 intraperitoneal filleur et al. 2003 flynn et al. 2004; de jonge et al. 2005; ocker et al. 2005; sorensen et al. 2003; verma et al. 2003; urban-klein et al. 2005; yin et al. 2005 intramuscular golzio et al. 2005 intratracheal lomas-neira et al. 2005 perl et al. 2005 intranasal bitko et al. 2005 zhang et al. 2004a , b subretinal reich et al. 2003 intraocular herard et al. 2005 intradermal kim et al. 2005 subcutaneous yano et al. 2004 intrathecal dorn et al. 2004 luo et al. 2005 stereotactic injection to hypothalamus makimura et al. 2002 infusion into the ventricular system (brain) hassani et al. 2005; thakker et al. 2004 thakker et al. , 2005 intrathecal infusion using miniosmotic pump dorn et al. 2004 in situ perfusion/intravenous (pancreatic islet) bradley et al. 2005a , b intracardiac bollerot et al. 2006 intratumoral bertrand et al. 2002 ito et al. 2005; leng and local injection and electroporation (mouse joint) schiffelers et al. 2005 in vivo studies in xenografted mice have demonstrated that the i.p. injection of pei-complexed sirnas, but not of naked sirnas, resulted in the delivery of intact sirna molecules into the subcutaneous tumors, leading to antitumorigenic effects when targeting for example the receptor her-2 in s.c. ovarian carcinoma xenografts (urbanklein et al. 2005) or the growth factors pleiotrophin (ptn, s.c. glioblastoma) (grzelinski et al. 2006) or vegf (s.c. prostate carcinoma; hobel et al., unpublished data) . likewise, intrathecal delivery of pei-complexed specific sirnas led to successful ptn targeting and tumor inhibition in an ortotopic glioblastoma mouse model (grzelinski et al. 2006) , or to the knockdown of the nmethyl-d-aspartate (nmda) receptor subunit protein nr2b in a rat model (tan et al. 2005) . pei complexation of sirnas has also been employed in antiviral therapy studies (ge et al. 2004; geisbert et al. 2006) . furthermore, pei/ sirna complexes are a good example for the introduction of chemical modifications to enhance tissue specificity and in vivo biocompatibility, to reduce immunogenicity and toxicity and to increase sirna delivery through improved endocytosis and intracellular sirna release. this includes full deacetylation of pei (thomas et al. 2005) , the introduction of novel low molecular weight peis (werth et al. 2006) , and the coupling of pei to other macromolecules like polyethylene glycol (peg), either alone (mao et al. 2006) or in combination with a ligand for tissuespecific targeting (rgd peptide for the recognition of tumor vasculature; schiffelers et al. 2004 ). alternatively, the goals of increased systemic sirna stability and serum half-life have also been achieved bradley et al. 2005a, b; duxbury et al. 2004; giladi et al. 2003; heidel et al. 2004; hino et al. 2006; klein et al. 2003; lewis et al. 2002; liang et al. 2005; matsui et al. 2005; merl et al. 2005; sato et al. 2005; song et al. 2003; tompkins et al. 2004; zender et al. 2003 chemically modified, naked braasch et al. 2004 elmen et al. 2005; soutschek et schiffelers et al. 2004 through extensive chemical modifications of the sirna strands including the introduction of phosphorothioate (braasch et al. 2003 (braasch et al. , 2004 , 4′ thioribose (dande et al. 2006) or methylene linkages between positions 2′ and 4′ (locked nucleic acids, lnas; braasch et al. 2003; elmen et al. 2005) , or multiple 2′ modifications (amarzguioui et al. 2003; harborth et al. 2003; holen et al. 2002 holen et al. , 2003 . additionally, chemical conjugation of sirnas, e.g., to cholesterol (soutschek et al. 2004) or to a protamin-antibody fusion protein led to enhanced efficacy and specificity in tissue uptake. for other strategies, refer to table 3 . rnai has already proven to be a very efficient and specific method for the knockdown of physiologically or pathologically relevant genes of interest. notably, this also applies to so-called "nondruggable" genes, thus opening new therapeutic avenues. still, the therapeutic applicability and success of sirnas will largely depend on their efficient and safe in vivo delivery avoiding unwanted side effects. reflecting the high relevance of rnai, many studies have been published or are ongoing, which will finally allow to identify optimal strategies based on already promising results. this also refers to the first clinical trials, which are completed or ongoing. most likely, one major advantage of formulated, modified, or unmodified sirnas for gene knockdown will be their "double specificity", i.e., the combination of a high target gene specificity through optimal sirna sequences and an at least somewhat increased target organ specificity through sophisticated delivery vehicles like liganded nanocarriers. transcriptional and phenotypic comparisons of ppara knockout delivery of unmodified bioactive ribozymes by an rnastabilizing polyethylenimine (lmw-pei) efficiently down-regulates gene expression tolerance for mutations and chemical modifications in a sirna ex vivo and in vivo delivery of anti-tissue factor short interfering rna inhibits mouse pulmonary metastasis of b16 melanoma cells controlled release of small interfering rna targeting midkine attenuates intimal hyperplasia in vein grafts progress towards in vivo use of sirnas the proton sponge: a trick to enter cells the viruses did not exploit role for a bidentate ribonuclease in the initiation step of rna interference comparison of antisense oligonucleotides and sirnas in cell culture and in vivo inhibition of respiratory viruses by nasally administered sirna c-versus n-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of dna polyplexes melittin analogs with high lytic activity at endosomal ph enhance transfection with purified targeted pei polyplexes widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine rna interference in mammalian cells by chemicallymodified rna biodistribution of phosphodiester and phosphorothioate sirna successful incorporation of short-interfering rna into islet cells by in situ perfusion gene silencing in the endocrine pancreas mediated by shortinterfering rna induction of an interferon response by rnai vectors in mammalian cells novel cationic cardiolipin analoguebased liposome for efficient dna and small interfering rna delivery in vitro and in vivo structural domains in rnai improving rna interference in mammalian cells by 4′-thio-modified small interfering rna (sirna): effect on sirna activity and nuclease stability when used in combination with 2′-o-alkyl modifications reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of small-interfering rnas rad51 sirna delivered by hvj envelope vector enhances the anticancer effect of cisplatin expression profiling reveals off-target gene regulation by rnai sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna efficient new cationic liposome formulation for systemic delivery of small interfering rna silencing tumor necrosis factor alpha in experimental arthritis modification of professional antigen-presenting cells with small interfering rna in vivo to enhance cancer vaccine potency inhibition of hepatitis b virus replication in vivo by nucleoside analogues and sirna therapeutic epha2 gene targeting in vivo using neutral liposomal small interfering rna delivery intraperitoneal delivery of liposomal sirna for therapy of advanced ovarian cancer small interfering rna targeting raf-1 inhibits tumor growth in vitro and in vivo silencing of cxcr4 blocks breast cancer metastasis down-regulation of apoptosis mediators by rnai inhibits axotomy-induced retinal ganglion cell death in vivo argonaute2 is the catalytic engine of mammalian rnai in vivo gene silencing (with sirna) of pulmonary expression of mip-2 versus kc results in divergent effects on hemorrhageinduced, neutrophil-mediated septic acute lung injury an efficient intrathecal delivery of small interfering rna to the spinal cord and peripheral neurons cationic lipids enhance sirna-mediated interferon response in mice in vitro and in vivo suppression of gjb2 expression by rna interference reducing hypothalamic agrp by rna interference increases metabolic rate and decreases body weight without influencing food intake vivo potentialities of ews-fli-1 targeted antisense oligonucleotidesnanospheres complexes influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/sirna polyplexes single-stranded antisense sirnas guide target rna cleavage in rnai sequencespecific suppression of mdr1a/1b expression in mice via rna interference rna interference in adult mice targeting 2a protease by rna interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes atelocollagen-mediated synthetic small interfering rna delivery for effective gene silencing in vitro and in vivo sirna-induced caveolin-1 knock-down in mice increases lung vascular permeability via the junctional pathway activity of stabilized short interfering rna in a mouse model of hepatitis b virus replication potent and persistent in vivo anti-hbv activity of chemically modified sirnas intravesical administration of small interfering rna targeting plk-1 successfully prevents the growth of bladder cancer atp requirements and small interfering rna structure in the rna interference pathway variants of bcl-2 specific sirna for silencing antiapoptotic bcl-2 in pancreatic cancer systemic delivery of rafsirna using cationic cardiolipin liposomes silences raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer silencing of fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis intravenous delivery of antirhoa small interfering rna loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer tumor-targeting nanoimmunoliposome complex for short interfering rna delivery biochemical identification of argonaute 2 as the sole protein required for rna-induced silencing complex activity small interfering rna (sirna) targeting vegf effectively inhibits ocular neovascularization in a mouse model purified argonaute2 and an sirna form recombinant human risc a novel sirnalipoplex technology for rna interference in the mouse vascular endothelium rna interference in the mouse vascular endothelium by systemic administration of sirna-lipoplexes for cancer therapy gene silencing in rat-liver and limb grafts by rapid injection of small interference rna cancer sirna therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle effects of treatment with small interfering rna on joint inflammation in mice with collagen-induced arthritis suppression of ocular neovascularization with sirna targeting vegf receptor 1 egf receptor-targeted synthetic double-stranded rna eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded sirnas is sequence-dependent and requires endosomal localization cationic liposome-mediated delivery of sirnas in adult mice activation of the interferon system by short-interfering rnas rna interference targeting fas protects mice from fulminant hepatitis antibody mediated in vivo delivery of small interfering rnas via cellsurface receptors gene silencing by systemic delivery of synthetic sirnas in adult mice therapeutic silencing of an endogenous gene by systemic administration of modified sirnas exploring rna interference as a therapeutic strategy for renal disease gene silencing in primary and metastatic tumors by small interfering rna delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases a small interfering rna targeting vascular endothelial growth factor as cancer therapeutics efficient delivery of small interfering rna to bone-metastatic tumors by using atelocollagen in vivo gene knockdown with intrathecal sirna of nmda receptor nr2b subunit reduces formalin-induced nociception in the rat the influence of polymer structure on the interactions of cationic polymers with dna and morphology of the resulting complexes neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral rna interference sirna-mediated knockdown of the serotonin transporter in the adult mouse brain full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung protection against lethal influenza virus challenge by rna interference in vivo rnai-mediated gene-targeting through systemic application of polyethylenimine (pei)-complexed sirna in vivo small interfering rnas directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells a low molecular weight fraction of polyethylenimine (pei) displays increased transfection efficiency of dna and sirna in fresh or lyophilized complexes in vivo delivery of caspase-8 or fas sirna improves the survival of septic mice first clinical data on rnai single-walled carbon nanotubesmediated in vivo and in vitro delivery of sirna into antigenpresenting cells antitumor activity of small interfering rna/cationic liposome complex in mouse models of cancer silencing heat shock factor 1 by small interfering rna abrogates heat shockinduced cardioprotection against ischemia-reperfusion injury in mice caspase 8 small interfering rna prevents acute liver failure in mice hydroporation as the mechanism of hydrodynamic delivery small interfering rna targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis small interfering rnas targeting mutant k-ras inhibit human pancreatic carcinoma cells growth in vitro and in vivo rnai-mediated gene silencing in nonhuman primates acknowledgments the author's work reported herein was supported by grants from the deutsche forschungsgemeinschaft (ai 24/5-1 and forschergruppe nanohale, ai 24/6-1) and by the deutsche krebshilfe. key: cord-012098-1nxws0dk authors: van den brink, joost; de vries, ronald p. title: fungal enzyme sets for plant polysaccharide degradation date: 2011-07-23 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3473-2 sha: doc_id: 12098 cord_uid: 1nxws0dk enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. these polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. to enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. the variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. this mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families. plant polysaccharides have applications in many industrial sectors, such as biofuel, pulp and paper, and food and feed. cellulose, hemicelluloses, and pectin are the main components of plant cell walls representing up to 70% of the biomass (jorgensen et al. 2007 ). of the three, cellulose is the least complex polymer with a linear structure of β-1,4linked d-glucose residues. the long glucose chains are tightly bundled together in microfibrils and are noncovalently linked together by hemicelluloses (kolpak and blackwell 1976; carpita and gibeaut 1993) . hemicelluloses are classified according to the main sugar in the backbone of the polymer, i.e., xylan (β-1,4linked d-xylose), mannan (β-1,4-linked d-mannose), or xyloglucan (β-1,4-linked d-glucose). the backbone of hemicelluloses has many branches composed of monomers such as d-galactose, d-xylose, l-arabinose, and d-glucuronic acid. the precise composition of hemicellulose is strongly dependent on the plant species and tissue (scheller and ulvskov 2010) . for instance, hard wood xylans often have d-glucuronic acid attached to their backbone, whereas larabinose is the most common branching residue in cereal xylans . moreover, hemicelluloses are often acetylated and to a lesser extent ester-linked with feruloyl or p-coumaroyl residues (ebringerova et al. 1990; xu et al. 2010) . pectin is less prominently present in most plant biomass compared to cellulose and hemicellulose. however, some plant biomass types (e.g., citrus peels) are very rich in pectin (angel siles lopez et al. 2010; ridley et al. 2001; grohmann and bothast 1994) . the backbone of pectin consists mainly of alpha-1,4-linked d-galacturonic acid residues that can be methyl-esterified or substituted with acetyl groups. pectins are classified in three general groups, homogalacturonan (linear polymer), xylogalacturonan (branched by β-1,3-linked d-xylose), and rhamnogalacturonan (ridley et al. 2001; wong 2008; caffall and mohnen 2009) . the latter polysaccharide is the most complex pectin structure. its backbone consists of alternating l-rhamnose and d-galacturonic acid residues, while branches with β-1,4-linked d-galactose and different α-linked l-arabinose residues are connected to the l-rhamnose residues (ridley et al. 2001; wong 2008) . in nature, fungi play a central role in the degradation of plant biomass. plant-biomass-degrading fungi produce an extensive set of carbohydrate-active enzymes specifically dedicated to degrade plant polysaccharides. however, these sets differ between fungal species. for instance, trichoderma reesei has a highly efficient set of enzymes involved in cellulose degradation (martinez et al. 2008; kubicek et al. 2011) , while aspergillus species produce many enzymes to degrade pectin (martens-uzunova and schaap 2009). the industrial importance of polysaccharide-degrading enzymes and the availability of many fungal genomes have strongly deepened our understanding of fungal biodiversity with respect to plant cell wall degradation. this minireview will give an overview of the latest developments and insights into fungal enzymes involved in plant polysaccharide degradation. carbohydrate-active enzymes can be organized in different families based on amino acid sequence of the structurally related catalytic modules (www.cazy.org) (cantarel et al. 2009; henrissat 1991) . fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase (gh) families, three carbohydrate esterase (ce) families, and six polysaccharide lyase (pl) families coutinho et al. 2009 ). even though enzymes within the same family share sequence similarity, some families can contain multiple activities. for example, gh5 contains many catalytic activities, including exoglucanases, endoglucanases, and endomannanases (dias et al. 2004 ). in addition, a specific enzyme activity can be present in several cazy families. this is important for efficient degradation of plant polysaccharides as enzymes of each family have often complementary substrate specificity. for instance, endoxylanases in gh10 have lower substrate specificity and can degrade xylan backbones with many substitutions, while gh11 endoxylanases have higher substrate specificity with preference for unsubstituted xylan chains (pollet et al. 2010; biely et al. 1997) . annotation of carbohydrate-active enzymes has been done for many fungal genomes (pel et al. 2007; espagne et al. 2008; battaglia et al. 2011; ohm et al. 2010; martinez et al. 2004; martinez et al. 2008) . as an illustration, table 1 shows a comparison of carbohydrate-active enzymes involved in plant polysaccharide degradation of 13 fungal genomes, including industrial fungi such as aspergillus oryzae, aspergillus niger, penicillium chrysogenum, t. reesei, and saccharomyces cerevisiae. most apparent from this table is the correlation between habitat and the amount of carbohydrate-active enzymes. for example, the saccharomycete s. cerevisiae does not require extracellular enzymes for polysaccharide degradation to survive in its natural niches like surfaces of rotting fruits (liti et al. 2009; cherry et al. 1997) . this fungus has therefore hardly any carbohydrate-active enzymes. another saccharomycete pichia stipitis can be found, among other places, in the guts of termites that inhabit and degrade white-rotted hardwood (jeffries et al. 2007 ). the genome of this fungus contains only a few β-glucosidases and β-mannosidases to degrade glucan and mannan oligosaccharides, which are present in the termite guts (jeffries et al. 2007) . in contrast to both saccharomycetes, the filamentous fungi in table 1 have a lifestyle involving degradation of plant biomass and feeding from plant polysaccharides. the genomes of these fungi therefore contain many more genes encoding carbohydrate-active enzymes. for example, the saprobe a. niger has 178 putative gh enzymes, nine putative ce enzymes, and 13 putative pl enzymes involved in plant biomass degradation ). however, there is also variation within this group of fungi. for instance, aspergilli have a large number of enzymes involved in pectin degradation, in contrast to the cellulose-degrading specialist t. reesei and the lignin-degrader phanerochaete chrysosporium. the zygomycete rhizopus oryzae has a different set of carbohydrate-active enzymes compared to the other filamentous fungi . for instance, the difficulty of this fungus to grow on xylan substrates is reflected in its absence of genes required for xylan degradation. this fungus has therefore also been described as a fast grower on easily accessible and digestible substrates (richardson 2009 ). to have a better impression of the latest developments regarding fungal carbohydrate-active enzymes, the following sections will discuss the enzymes needed for each of the main polysaccharides: cellulose, hemicellulose, and pectin. furthermore, as an illustration of a fungal enzyme set, each section will show the genes encoding characterized and putative polysaccharide-degrading enzymes of a. niger. cellulose degradation requires three classes of enzymes, β-1,4-endoglucanases (egl), exoglucanases/cellobiohydrolases (cbh), and β-glucosidase (bgl), which are divided over eight gh families ( fig. 1 ; vlasenko et al. 2010; de vries et al. 2011) . for example, a. niger has five egls within gh families 5 and 12, four cbhs in families 6 and 7, and 13 bgls in families 1 and 3 (table 2 ). in comparison, one of the most efficient cellulose-degrading fungi t. reesei has five characterized egls within gh families 5, 7, 12, and 45, two highly expressed cbhs in families 6 and 7, and two characterized bgls in families 1 and 3 (martinez et al. 2008; kubicek et al. 2011) . although t. reesei does not have the biggest number of cellulases, its set of enzymes is very efficient in breaking down cellulose by acting synergistically (ward et al. 1993) . the family numbers are also used in the nomenclature of hydrolytic enzymes (henrissat et al. 1998) . for example, the first three letters of endoglucanase cel5a of t. reesei refer to its substrate cellulose, the number to glycoside hydrolase family 5 and the last capital letter indicates that this was the first enzyme reported of this family with this activity. this review will mainly refer to the original functionally based nomenclature. we will therefore use for instance egii instead of cel5a, based on the consideration that these names were mainly used in database depositions and give a clearer indication of function. egi (gh7) and egii (gh5) are the most abundantly produced of the five egls of t. reesei (foreman et al. 2003; vlasenko et al. 2010) . both enzymes have a carbohydrate-binding module which greatly enhances the efficiency in degradation of cellulose microfibrils by binding to cellulose (beckham et al. 2010; guillen et al. 2010 ). egiii, from gh12, is expressed at a lower level than egi and egii, but has a broad activity against cellulose, β-1,3-1,4-glucan, xyloglucan, and xylan (sprey and bochem 1993; eriksson et al. 2002) . two proteins from gh61 were expressed in the presence of cellulose and activity has been measured against β-glucan (saloheimo et al. 1997; foreman et al. 2003) . however, the enzymes within gh61 have recently shown not to be glycoside hydrolases (harris et al. 2010 ). although they are involved in improving the efficiency of degrading tightly packed cellulose microfibrils, the precise function of gh61 enzymes is still unknown (harris et al. 2010; vaaje-kolstad et al. 2010 ). the two highly expressed cbhs of t. reesei, cbhi and cbhii, belong to families 7 and 6, respectively. these are considered to work synergistically and have preference for the reducing or non-reducing end, respectively, of the cellulose chains (nutt et al. 1998) . cbhs are also considered to be important for the hydrolysis of the crystalline parts of cellulose (liu et al. 2011 ). these cbhs of t. reesei are highly sensitive to product inhibition, in particular by cellobiose, which might explain the need for a high amount of cbhs in an effective fungal cellulase enzyme mix (bezerra and dias 2005; holtzapple et al. 1990; kristensen et al. 2009 ). after endo-and exo-cleaving of cellulose, bgls belonging to gh families 1 and 3 degrade the remaining oligosaccharides to monomeric glucose. bgls are a group with very diverse properties and cellular location, although most bgls belong to gh3 and have a similar retaining mechanism (decker et al. 2001; saloheimo et al. 2002) . the two known bgls of t. reesei, bgi and bgii, are produced at low levels (reczey et al. 1998 ) and subject to strong product inhibition (chen et al. 1992) . these characteristics hinder the function of t. reesei for extensive in vitro saccharification of cellulose. therefore, in industrial applications, t. reesei cellulase mixtures are often supplemented with bgls from aspergilli, which are highly expressed and more glucose tolerant (decker et al. 2000; reczey et al. 1998; riou et al. 1998) . another strategy to convert cellulose into a sustainable product is to express bgls in the fermentation host like s. cerevisiae ha et al. 2011 ). this way, oligosaccharides like cellobiose are directly fermented into fuels or chemicals. hemicellulose, the second most abundant plant polysaccharide, is a group of complex structures composed of different residues with three kinds of backbones and many different branches (fig. 2) . the three backbones of the corresponding group of hemicelluloses are hydrolysed by a specific set of dedicated carbohydrate-active enzymes: β-1,4endoxylanase and β-1,4-xylosidase for xylan, xyloglucanactive β-1,4-endoglucanase and β-1,4-glucosidase for xyloglucan, and β-1,4-endomannanase and β-1,4-mannosidase for (galacto-) mannan (table 3 ) (de vries and visser 2001). the fungal β-1,4-endoxylanases belong to gh families 10 and 11 (polizeli et al. 2005) . similar to enzymes within families gh1, gh2, and gh5, endoxylanases of gh10 have a tim-barrel fold at their catalytic domain in contrast to gh11 which has a β-jelly roll structure at its catalytic domain (henrissat and bairoch 1993; pollet et al. 2010) . as a consequence, the two groups of endoxylanases differ from each other in substrate specificity (biely et al. 1997 ). gh10 endoxylanases have in general a broader substrate specificity then endoxylanases of family gh11. specifically, gh10 enzymes not only degrade linear chains of 1,4-linked dxylose residues, but also xylan backbones with a high degree of substitutions and smaller xylo-oligosaccharides (biely et al. 1997; vardakou et al. 2003; pollet et al. 2010 ). gh10 endoxylanases are therefore important for the complete degradation of substituted xylans. although, in principal, less accessory enzymes are required with a higher amount of gh10 endoxylanases, fungal genomes have no clear correlation between the amount of gh10 endoxylanases and number of accessory enzymes. the released xylo-oligosaccharides are degraded by βxylosidases. most fungal β-xylosidases belong to gh3 (mozolowski and connerton 2009), but several putative βxylosidases are assigned to gh43 (e.g., in penicillium herquei and a. oryzae (ito et al. 2003; machida et al. 2005) ). gh3 is a conserved family containing mainly bgls. as shown in t. reesei and bacteria, β-xylosidases of gh3 contain the conserved asp-311 residue but miss the other active-site asp-120 residue of β-glucosidases, which might explain the an18g03570 (bgl1), an07g09760, an08g08240, an11g00200, an14g01770, an17g00520, an03g05330, an06g02040, an15g01890, an11g06090, an15g04800 (dan et al. 2000; pel et al. 2007) the genes with names between brackets are biochemically characterized and their references are given in the last column xyloglucan-active endoglucanases, also referred to as xyloglucanases, belong to gh families 12 and 74 (grishutin et al. 2004 ). the main difference between enzymes of gh12 and gh74 is their retaining and inverting mechanism, respectively (gilbert et al. 2008) . the specific modes of action of the different xyloglucanases were recently elucidated (desmet et al. 2007; powlowski et al. 2009; yaoi et al. 2009; master et al. 2008) . for instance, in contrary to the gh74 xyloglucanase from t. reesei, the gh12 enzyme from a. niger does not cleave at branched glucose residues and prefers xylogluco-oligosaccharides containing more than six glucose residues with, at least, one non-branched glucose residue (master et al. 2008; desmet et al. 2007 ). endomannanases, involved in the degradation of mannan polysaccharides, belong to gh families 5 and 26. however, fungal endomannanases are predominately present in gh5. the gh5 endomannanases from a. niger and t. reesei both show substrate specificity towards manno-oligosaccharides with more than three d-mannose residues do et al. 2009 ). like many other fungal carbohydrateactive enzymes, some endomannanases have a carbohydratebinding module (mainly cbm1) which promotes the fig. 2 a-c schematic structure of three hemicelluloses, xylan, galacto(gluco)mannan, and xyloglucan, with hemicellulolytic enzymes. abf αarabinofuranosidase, afc α-fucosidase, agl α-1,4galactosidase, agu αglucuronidase, axe acetyl (xylan) esterase, axh arabinoxylan α-arabinofuranohydrolase, axl α-xylosidase, bxl β-1,4xylosidase, fae feruloyl esterase, lac β-1,4-galactosidase, man β-1,4-endomannanase, mnd β-1,4-mannosidase, xeg xyloglucan-active β-1,4endoglucanase, xln β-1, 4-endoxylanase the genes with names between brackets are biochemically characterized and their references are given in the last column association of the enzyme with the substrate (herve et al. 2010; pham et al. 2010; boraston et al. 2004 ). the released mannobiose and mannotriose are further degraded by β-1,4-mannosidases belonging to gh family 2 (ademark et al. 2001) . to completely degrade hemicellulose, all substitutions on the hemicellulose backbones have to be released. this requires at least nine different enzyme activities divided over at least 12 gh families and four ce families (table 4) . l-arabinose, a common residue in hemicellulose, is cleaved from arabinose-substituted xyloglucan and (arabino-) xylan by α-arabinofuranosidases and arabinoxylan arabinofuranohydrolases. fungal α-arabinofuranosidases are mainly found in gh families 51 and 54, although some bifunctional enzymes from gh3 and gh43 were described to have αarabinofuranosidase activity (saha 2000; ravanal et al. 2010) . the difference in substrate specificity between gh51 and gh54 enzymes is illustrated by the two main arabinofuranosidases of a. niger. abfa (gh51) releases larabinose from arabinan and sugar beet pulp, while abfb (gh54) also releases l-arabinose from xylan (de vries and visser 2001). arabinofuranosidases within gh54 are also described to have a carbohydrate-binding module (cbm 42) with specific binding to arabinofuranose side chains of hemicellulose (miyanaga et al. 2004; miyanaga et al. 2006 ). the arabinoxylan arabinofuranohydrolases from gh62 act specifically against the α-1,2or α-1,3-linkage of the larabinose residues of arabinoxylan (verbruggen et al. 1998 ), but are also sensitive to the substitutions of adjacent d-xylose residues. for instance, axha from a. niger is not able to release arabinobiose from xylan or substituted larabinose from d-xylose residues adjacent to d-glucuronic acid residues (verbruggen et al. 1998; sakamoto et al. 2011 ). d-xylose residues with α-linkages are released from the xyloglucan backbone by α-xylosidases. of two αxylosidases in aspergillus flavus, axli hydrolyzes xyloglucan oligosaccharides and axlii is most active on p-nitrophenyl α-l-xylose residues and does not hydrolyze xyloglucan (yoshikawa et al. 1993 (yoshikawa et al. , 1994 . α-xylosidases were suggested to belong to family gh31 based on genome analysis within aspergilli which was proven by overexpression in pichia pastoris (bauer et al. 2006; de vries et al. 2005) . this gh31 family contains mainly enzymes with α-glucosidase activity and has only a limited number of characterized α-xylosidases (de vries et al. 2005) . l-fucose residues in xyloglucan branches are released by α-fucosidases belonging to gh29 and gh95. several αfucosidases of plants have been identified to degrade xyloglucan (leonard et al. 2008; ishimizu et al. 2007; minic and jouanin 2006) . nevertheless, only a. niger, aspergillus nidulans, and penicillium multicolor have been reported to produce an α-fucosidase which release l-fucose residues similar to the fucose-linkages in xyloglucan (ajisaka et al. 1998; ajisaka and shirakabe 1992; bauer et al. 2006) . alpha-linked d-galactose residues are released from hemicellulose, e.g., xylan and galactomannans, by αgalactosidases belonging to gh27 and gh36. the αthe genes with names between brackets are biochemically characterized and their references are given in the last column galactosidases of gh27 and gh36 both act via a doubledisplacement mechanism and are considered to have a common evolutionary origin (rigden 2002) . some enzymes of gh27 also showed α-n-acetylgalactosaminidase activity and it is therefore argued that some gh27 α-galactosidases are not involved in hemicellulose degradation (kulik et al. 2010) . the gh36 α-galactosidases are often larger in size and are active against mono-and oligosaccharides, such as melibiose and raffinose (ademark et al. 2001) . the presence of terminal β-linked d-galactose residues in some hemicelluloses, e.g., xylan, xyloglucan, and galactoglucomannans, suggested that β-galactosidases (gh2 and gh35) also play a role in degradation of hemicelluloses (sims et al. 1997) . this was proven by the increased release of dgalactose residues from wheat flour after supplementing laca of a. niger to an enzyme cocktail . bga1, a β-galactosidase of t. reesei, showed broad substrate specificity with also activity against polymeric galactans (gamauf et al. 2007 ). nevertheless, the fast majority of studies concerning β-galactosidases are focused on their activity against lactose. d-glucuronic acid residues from polymeric xylan are released by α-glucuronidases belonging to gh67 and the newly identified family gh115 (ryabova et al. 2009; chong et al. 2011) . like many other carbohydrate-active enzyme families, the difference between α-glucuronidases from both families lays in their substrate specificity. gh67 α-glucuronidases are active on short oligosaccharides, while some of the gh115 α-glucuronidases are active on polymeric xylan (chong et al. 2011; tenkanen and siika-aho 2000) . the gh115 α-glucuronidase from pichia stipitis showed however higher activity against short oligosaccharides, which corresponds with its ability to degrade oligosaccharides within their environment (kolenova et al. 2010) . remarkably, gh67 α-glucuronidases are found exclusively within ascomycetes, while gh115 α-glucuronidases are present in ascomycetes and basidiomycetes (chong et al. 2011) . acetyl residues from xylan chains are released by acetylxylan esterases belonging to ce families 1, 4, 5, and 16 (biely et al. 2011) . the presence of acetylxylan esterases is essential for efficient degradation of the xylan backbone by endoxylanases. for instance, only in the presence of a. niger acetylxylan esterase, birchwood xylan can be degraded by the three endoxylanases and the βxylosidase of a. niger (kormelink et al. 1993) . the main difference between the ce families is their preference for hydrolyzing the different o-linked acetyl groups. ce families 1, 4, and 5 have a strong preference for 2-olinked residues, the most common linkage in hemicellulose, while ce16 prefers 3-o-and 4-o-linked residues (li et al. 2008; biely et al. 2011 ). there has also been a description of acetyl esterase of a. oryzae active against acetyl residues attached to galactomannan chains (tenkanen et al. 1995) . however, the gene encoding this specific enzyme has not yet been identified nor characterized in other fungi. p-coumaric acid and ferulic acid, the two cinnamic acids present in xylan, are removed by feruloyl/p-coumaroyl esterases. most of these esterases have not been assigned to ce families. however, several classifications have been reported for these enzymes based on sequence similarity and substrate specificity (crepin et al. 2004; olivares-hernandez et al. 2010; benoit et al. 2008) . one particular group of esterases, belonging to aspergillus and penicillium sp., has preference for substrates with methoxy substituents (koseki et al. 2009; kroon et al. 1997) . for example, faea of a. niger prefers substrates with a methoxy group at position three, such as ferulic acid (benoit et al. 2007; kroon et al. 1997) . faeb of a. niger belongs to another group of esterases with preference for substrates containing one or two hydroxyl substitutions, such as p-coumaric acid (de vries et al. 1997; koseki et al. 2009; kroon et al. 1997 ). the degradation of pectin backbones (fig. 3) requires two classes of enzymes: glycoside hydrolases and polysaccharide lyases (pl; table 5 ). a large part of the fungal glycoside hydrolases involved in the degradation of the pectin backbone belongs to gh family 28 (martens-uzunova and schaap 2009). these gh28 enzymes can be divided in groups according to the specific pectin region they attack: endo-and exo-polygalacturonase (gh28) cleave the backbone of the smooth regions, while the more intricate, hairy regions are further attacked by endo-and exo-rhamnogalacturonase (gh28), xylogalacturonase (gh28), α-rhamnosidases (gh78), unsaturated glucuronyl hydrolases (gh88), and unsaturated rhamnogalacturonan hydrolases (gh105). endo-and exo-polygalacturonases of gh28 in general cleave the α-1,4-glycosidic bonds between the α-galacturonic acids. the genome of a. niger contains seven endopolygalacturonases, each of them exhibiting distinct kinetic properties, substrate methylation sensitivity and mode of action (martens-uzunova and schaap 2009; benen et al. 2000; bussink et al. 1991; parenicova et al. 1998 parenicova et al. , 2000a . for instance, although the structures of pgai and pgaii are highly similar, only pgai has enzyme processivity due to a narrower substrate binding cleft and the presence of an arginine at position 96 (van santen et al. 1999; van pouderoyen et al. 2003) . the genome of a. niger contains four potential exopolygalacturonases: pgax, pgxa, pgxb, and pgxc (martens-uzunova et al. 2006) . of these, pgxb prefers homogalacturonan as a substrate, while pgxc has high activity against homogalacturonan and xylogalacturonan (martens-uzunova et al. 2006 ). pgxa has a low activity against homogalacturonan and is more active against xylogalacturonan. therefore, pgxa very likely represents a new kind of exoxylogalacturanase (martens-uzunova et al. 2006 ). an endo-acting xylogalacturanase, xgha, was already described to specifically degrade the xylogalacturonan present in the hairy regions (van der vlugt-bergmans et al. 2000) . also within r. oryzae, 15 of the 18 putative gh28 polygalacturonases showed different substrate specificity for polygalacturonan, trigalacturonan, and digalacturonan, and their activity against polygalacturonan ranged from less than 1 to 200 μmol/min/mg protein (mertens and bowman 2011) . these different characteristics between polygalacturonases of a. niger and r. oryzae shows the requirement for a large number of isoenzymes to concertedly act on complex plant polysaccharides. another important group of gh28 are the rhamnogalacturonan hydrolases, which employ either an endo-or exolytic mechanism to cleave the α-1,2-glycosidic bonds formed between d-galacturonic acid and l-rhamnose residues in the hairy regions (kofod et al. 1994; suykerbuyk et al. 1995) . a. niger has two characterized (rhga and rhgb) and four putative endorhamnogalacturonanases (martens-uzunova and schaap 2009; suykerbuyk et al. 1997) . the pattern of reaction products produced after degradation of modified hairy regions with either rhga or rhgb is quite different, suggesting that each enzyme acts on a structurally different region of the substrate (suykerbuyk et al. 1997) . exorhamnogalacturonases release d-galacturonic acid residues from the non-reducing end of rhamnogalacturonan chains but not from homogalacturonans (mutter et al. 1998) . sequence analysis of the a. niger genome indicates the presence of three genes, rgxa, rgxb, and rgxc, encoding putative exorhamnogalacturonases (martens-uzunova et al. 2006) . hydrolysis of the pectin backbone also requires enzymes from other gh families: α-rhamnosidases (gh78), unsaturated glucuronyl hydrolases (gh88), and unsaturated rhamnogalacturonan hydrolases (gh105). as several of these enzymes and families have only recently been described, little biochemical characterization has been performed on them (see table 5 for the putative genes of a. niger). for example, characterization of unsaturated glucuronyl hydrolases and unsaturated rhamnogalacturonan hydrolases is lacking in fungi, although several putative enzymes have been identified. pectin and pectate lyases both cleave, via a βelimination mechanism, the α-1,4-linked d-galacturonic acid residues within the smooth regions of pectin . a comparison between the structures of pectin and pectate lyases has indicated that both lyases most likely descended from a common ancestor enzyme (mayans et al. 1997; vitali et al. 1998 ). however, both types of lyases have important differences in their active site. as a consequence, pectin lyases attack preferentially heavily methyl-esterified substrates and have their optimum ph around 5.5 (mayans et al. 1997) . in contrast, pectate lyases favor lower degrees of esterification, have their optimum fig. 3 a-c schematic structures of three pectins, rhamnogalacturonan i, homogalacturonan, xylogalacturonan, with pectinolytic enzymes. abf αarabinofuranosidase, abn endoarabinanase, abx exoarabinanase, bxl β-1,4-xylosidase, fae feruloyl esterase, gal β-1,4-endogalactanase, lac βgalactosidase, pel pectin lyase, ply pectate lyase, pga endopoly-galacturonase, pgx exo-polygalacturonase, pme pectin methyl esterase, rgae rhamnogalacturonan acetyl esterase, rgl rhamnogalacturonan lyase, rhg endorhamnogalacturonase, rgx exorhamnogalacturonase, xgh endoxylo-galacturonase, xgx exoxylogalacturonase. α-rhamnosidase (rha), unsaturated rhamnogalacturonase (urh), and unsaturated glucuronyl hydrolase (ugh) are not depicted in this figure ph around 8.5, and require ca 2+ for their activity (mayans et al. 1997) . currently, all characterized pectin lyases belong to the pl1 family, while the fungal pectate lyases belong to pl1, pl3, and pl9. as an example, six pectin lyases and only one pectate lyase have been identified and partially characterized in a. niger harmsen et al. 1990; gysler et al. 1990 ). in contrast, a. nidulans has only two pectin lyases, five identified pectate lyases, and another six putative pectate lyases bauer et al. 2006) , suggesting significant differences between these fungi ). rhamnogalacturonan lyases differ substantially in their structure from pectin and pectate lyases, and cleave within the hairy regions of pectin. this group of lyases belongs to two families, pl4 and pl11, where pl4 lyases have a much lower optimum ph then pl11 lyases (jensen et al. 2010 ). the pl4 rhamnogalacturonan lyase from aspergillus aculeatus showed that cleavage preferably occurs four residues from l-rhamnose at the reducing end and is severely affected by the presence of acetyl groups in the backbone of rhamnogalacturonan (mutter et al. 1998; . therefore, the cooperative action of rhamnogalacturonan acetyl-esterases is required for efficient degradation . the pectin structures xylogalacturonan and rhamnogalacturonan also require accessory enzymes to remove the side chains and provide access for the main-chain hydrolysing pectinolytic enzymes. of these, α-arabinofuranosidases (gh51 and gh54), β-galactosidases (gh2 and gh35), and β-xylosidases (gh3 and gh43) are also needed for hemicellulose degradation, while endoarabinanases (gh43), exoarabinanases (gh93), β-endogalactanases (gh53), and several the genes with names between brackets are biochemically characterized and their references are given in the last column a exoarabinanase has only been identified in a. niger isolate atcc1015 esterases (ce8, ce12, and ce13) are specific for pectin degradation (martens-uzunova and schaap 2009). many industrial processes make use of relative easy accessible sugar residues within cellulose or other plant polysaccharides. in industrial processes, the monomeric sugars within these structures are released from the plant material by a limited set of enzymes, utilizing only a small fraction of the available biodiversity of fungal enzymes. for instance, a minimal mixture of cellobiohydrolases (cbhi and cbhii) and endoglucanase (egi) from t. reesei and βglucosidase from a. niger is sufficient to efficiently degrade pure cellulose (sternberg et al. 1977; rosgaard et al. 2007a ). exploitation of the available sugars within the intricate fractions of plant biomass involves a more elaborate process. for example, plant polysaccharides have to be separated from the present lignin compounds by chemical, physical, or biological pretreatment (hendriks and zeeman 2009; martinez et al. 2009 ). and, as described in the previous sections, releasing the sugars from the different plant polysaccharides requires a more complex set of carbohydrate-active enzymes. in addition to a number of cellulases, efficient enzymatic hydrolysis of pretreated plant biomass also needs synergistic activity of, at least, several endoxylanases, β-xylosidase, α-arabinofuranosidase, and acetyl esterase (kormelink et al. 1993; alvira et al. 2011; gao et al. 2010 ). the precise mixture of hydrolytic enzymes also depends on the method of pretreatment and type of plant substrate (rosgaard et al. 2007b; saha and cotta 2010) . nearly complete hydrolysis of specific xylan and pectin fractions can still be achieved with a relative small sets of enzymes , but this is not the case for more crude biomass fractions. for this reason, a better understanding of plant polysaccharide degradation will help to design an enzyme mixture which can efficiently degrade a wide range of substrates. the division of carbohydrate-active enzymes into different families based on modules of amino acid conservation (www. cazy.org) can help with a better understanding of the enzymatic repertoire of different fungi. in recent years, new families have been described and new activities have been assigned to known families (martens-uzunova et al. 2006; cantarel et al. 2009; li et al. 2008; chong et al. 2011; harris et al. 2010 ). nevertheless, more knowledge is required to fully profit from this large database of information. the biggest limitation is the low coverage of biochemical information on specific enzyme classes. this is illustrated by the small amount of enzymatically characterized proteins that has been added to the cazy database in the last decade in contrast to the large quantity of putative carbohydrate-active enzymes of sequenced fungal genomes (cantarel et al. 2009 ). as a consequence, some enzyme activities are putatively assigned to families, but do not have biochemical support. for example, none of the many putative fungal unsaturated glucuronyl hydrolases (gh88) and unsaturated rhamnogalacturonan hydrolases (gh105) have been biochemically characterized. another challenge is the large carbohydrate-active enzyme families containing enzymes with different enzyme classes. with increasing biochemical information, some families like gh2, gh3, gh5, gh28, or gh43 could be split up in smaller and better defined subfamilies according to their hydrolytic function. this will allow a better predictive value of future fungal genome annotations, and thereby 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family of diverged genes the structure, function, and biosynthesis of plant cell wall pectic polysaccharides structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth purification and characterization of two extracellular beta-glucosidases from trichoderma reesei botstein d (1997) genetic and physical maps of saccharomyces cerevisiae the alpha-glucuronidase agu1 from schizophyllum commune is a member of a novel glycoside hydrolase family (gh115) post-genomic insights into the plant polysaccharide degradation potential of aspergillus nidulans and comparison to aspergillus niger and aspergillus oryzae functional classification of the microbial feruloyl esterases the fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization cloning, expression, characterization, and nucleophile identification of family 3, aspergillus niger beta-glucosidase the faea genes from aspergillus niger and aspergillus tubingensis encode ferulic acid esterases involved in degradation of complex cell wall polysaccharides differential expression of three alphagalactosidase genes and a single beta-galactosidase gene from aspergillus niger synergy between enzymes from aspergillus involved in the degradation of plant cell wall polysaccharides expression profiling of pectinolytic genes from aspergillus niger the beta-1,4-endogalactanase a gene from aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions regulation of the alpha-glucuronidaseencoding gene (agua) from aspergillus niger the aspergillus niger faeb gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds the value of genome sequences in the rapid identification of novel genes encoding specific plant cell wall degrading enzymes fungal degradation of plant oligo-and polysaccharides. in: grunwald p (ed) carbohydrate modifying enzymes and microorganisms. pan stanford publishing pte beta-glucosidase multiplicity from aspergillus tubingensis cbs 643.92: purification and characterization of four beta-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance cloning and expression of a member of the aspergillus niger gene family encoding alpha-galactosidase an investigation of the substrate specificity of the xyloglucanase cel74a from hypocrea jecorina insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of cellvibrio mixtus mannosidase 5a cloning, expression in pichia pastoris, and characterization of a thermostable gh5 mannan endo-1,4-betamannosidase from aspergillus niger bk01 functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium prevotella bryantii b14 structural features of a water-soluble l-arabino-d-xylan from rye bran a model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase i (cel7a) and endoglucanase i (cel7b) of trichoderma reesei the genome sequence of the model ascomycete fungus podospora anserina molecular cloning, expression and structure of the endo-1,5-alpha-l-arabinase gene of aspergillus niger cloning and characterization of the abfb gene coding for the major alpha-l-arabinofuranosidase (abfb) of aspergillus niger cloning of the aspergillus niger gene encoding alpha-l-arabinofuranosidase a transcriptional regulation of biomass-degrading enzymes in the filamentous fungus trichoderma reesei the genome sequence of the filamentous fungus neurospora crassa characterization of the bga1-encoded glycoside hydrolase family 35 beta-galactosidase of hypocrea jecorina with galacto-beta-d-galactanase activity hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from aspergillus niger and aspergillus tubingensis two cellobiohydrolase-encoding genes from aspergillus niger require d-xylose and the xylanolytic transcriptional activator xlnr for their expression how the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation specific xyloglucanases as a new class of polysaccharide-degrading enzymes pectin rich residues generated by processing of citrus fruits, apples, and sugar beets carbohydratebinding domains: multiplicity of biological roles isolation and structure of the pectin lyase d-encoding gene from aspergillus niger engineered saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation cloning and expression of a second aspergillus niger pectin lyase gene (pela): indications of a pectin lyase gene family in a. niger stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family eglc, a new endoglucanase from aspergillus niger with major activity towards xyloglucan pretreatments to enhance the digestibility of lignocellulosic biomass a classification of glycosyl hydrolases based on amino acid sequence similarities new families in the classification of glycosyl hydrolases based on amino acid sequence similarities a scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects inhibition of trichoderma reesei cellulase by sugars and solvents a novel alpha1,2-l-fucosidase acting on xyloglucan oligosaccharides is associated with endo-beta-mannosidase xylosidases associated with the cell surface of penicillium herquei ifo 4674 genome sequence of the 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factors in high-solids enzymatic hydrolysis of lignocellulose methyl phenylalkanoates as substrates to probe the active sites of esterases comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of trichoderma the alphagalactosidase type a gene agla from aspergillus niger encodes a fully functional alpha-n-acetylgalactosaminidase saccharomyces cerevisiae cells secreting an aspergillus niger beta-galactosidase grow on whey permeate characterization of the aspergillus niger pelb gene: structure and regulation of expression identification of an arabidopsis gene encoding a gh95 alpha1,2-fucosidase active on xyloglucan oligo-and polysaccharides overproduction and characterization of xylanase b from aspergillus niger novel family of carbohydrate esterases, based on identification of the hypocrea jecorina acetyl esterase gene overcoming glucose repression in mixed sugar fermentation by co-expressing a cellobiose transporter and a beta-glucosidase in 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galactoglucomannan in pine kraft pulp an oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides genome sequencing and analysis of the filamentous fungus penicillium chrysogenum beta-xylosidase activity, encoded by xlnd, is essential for complete hydrolysis of xylan by aspergillus niger but not for induction of the xylanolytic enzyme spectrum the transcriptional activator xlnr regulates both xylanolytic and endoglucanase gene expression in aspergillus niger structural insights into the processivity of endopolygalacturonase i from aspergillus niger 68-a crystal structure of endopolygalacturonase ii from aspergillus niger and identification of active site residues by site-directed mutagenesis mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan enzymic degradation of sorghum glucuronoarabinoxylans leading to tentative structures the treedimensional structure of aspergillus niger pectin lyase b at 1.7-a resolution substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases enzymatic deconstruction of backbone structures of the ramified regions in pectins acetylation and characterization of spruce (picea abies) galactoglucomannans the crystal structure of a xyloglucan-specific endo-beta-1,4-glucanase from geotrichum sp. m128 xyloglucanase reveals a key amino acid residue for substrate specificity isolation of aspergillus flavus mo-5 producing two types of intracellular alpha-dxylosidases: purification and characterization of alpha-d-xylosidase i classification of some alpha-glucosidases and alpha-xylosidases on the basis of substrate specificity open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-012095-vzk2m91v authors: kraakman, norbertus j. r.; rocha-rios, jose; van loosdrecht, mark c. m. title: review of mass transfer aspects for biological gas treatment date: 2011-06-24 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3365-5 sha: doc_id: 12095 cord_uid: vzk2m91v this contribution reviews the mass transfer aspects of biotechnological processes for gas treatment, with an emphasis on the underlying principles and technical feasible methods for mass transfer enhancements. understanding of the mass transfer behavior in bioreactors for gas treatment will result in improved reactor designs, reactor operation, and modeling tools, which are important to maximize efficiency and minimize costs. various methods are discussed that show the potential for a more effective treatment of compounds with poor water solubility. biological gas treatment can be defined as the transformation of gaseous compounds to less harmful or more valuable products through the action of microorganisms. the treatment of gases using biological methods is rapidly gaining acceptance as a sustainable alternative to more conventional techniques such as chemical scrubbing, incineration, and adsorption where a pollutant is only transferred from the gas to other phase (adsorption) or where toxic by-products can be generated (chemical scrubbing and incineration). biological gas treatment is accepted as an economical and reliable air pollution control technology for treating gases with relatively low concentrations of pollutants (kennes and veiga 2001; groenestijn and van kraakman 2005; shareefden and singh 2005; estrada et al. 2011) . the growing emphasis on sustainability will even more stimulate the application of biological processes for gas treatment. the reactor lay-out of a biological gas treatment system is generally relatively simple, but the process of biological gas treatment involves a series of complex physical, chemical, and biological processes. many of these fundamental processes in biological gas treatment systems like mass transfer still require research (popat and deshusses 2010) . as a result, biological gas treatment systems are often built and operated without knowledge of the rate-limiting steps in the system (often resulting in scale-up problems) and design and operations are mainly based on empirical experience. the different biological techniques are traditionally classified in biofilters, biotrickling filters, and bioscrubbers. this classification has been enriched with different new configurations for biological gas treatment such as rotating bioreactor contactors, moving bed trickling filters, membrane bioreactors, bubble-tank bioreactors, airlift bioreactors, monolith bioreactors, twophase partitioning bioreactors, fungal biofilter, and mist bioreactors veiga 2001 and shareefden and singh 2005) . this diversification of the technology requires the knowledge of the rate-limiting steps in these systems in order to design, scale-up, and operate the most suitable system for a specific application. typically, a bioreactor for gas treatment is operating under mass transfer or kinetically limited conditions, and this review will focus on the mass transfer aspects of biological gas treatment techniques. gas-liquid mass transfer is an object of active research in many areas. efficient gas-liquid contacting is of great significance to many industrial applications, including biological gas treatment. when mass transfer is limited, the metabolic rate of the microorganisms decreases and the microorganisms may respond adversely to the resulting stress (lebrero et al. 2010) . a good understanding of the mass transfer behavior in bioreactors for gas treatment will result in improved modeling tools and more advanced reactor operations, which are important to maximize efficiency and minimize costs. traditionally, the enhancement of mass transfer in gasliquid contactors has been synonymous of an increase in power consumptions kreutzer et al. 2006) . unfortunately, in gas treatment where the treated gas flow can be as high as 10 5 m 3 h −1 and the footprint of the treatment equipment can be as large as a football field (kennes and veiga 2001), the energy required to obtain good interfacial contact area between gas and liquid in turbulent reactors can be immense. therefore, a structure (packed-bed) is used in laminar contactors (biofilters and biotrickling filters) to maximize the contact surface, but the lack of mixing in these systems leads to the presence of heterogeneities within the packed-bed (liu et al. 2005) . this means that new strategies to increase mass transfer in gas treatment operations while minimizing the power consumption need to be developed. this contribution reviews the mass transfer aspects of biotechnology for gas treatment in general terms, with an emphasis on the underlying principles and technically feasible methods for mass transfer enhancements of poorly water soluble compounds. biofilters and biotrickling filter are by far the most applied biotechnologies for gas treatment, but face important limitations when applied for the treatment of poor water soluble compounds due mass transfer limitations. this mini-review focuses on biofilters and biotrickling filters, but aims at making the findings available for other developing biotechnologies for gas treatment, which are rarely applied often due to high power consumption (shareefden and singh 2005) . understanding the rate-limiting steps in a system generates opportunities to optimize the design and operations of the system for a specific application. typically, the reaction in the reactor is operating under either mass transfer or kinetically limited conditions. in biological systems, such as biotrickling filters, bioscrubbers, and bubble-tank bioreactors with a mobile water phase that contains biomass, increasing the biomass concentration in the liquid phase would be a relatively simple way to determine whether the reactor is operating under mass transfer or kinetically limited conditions. when a change in the amount of pollutant removed per reactor volume (removal capacity) is seen at a sudden increased biomass concentration, the operation of the system is kinetically limited. another way to determine whether the reactor is operating under mass transfer or kinetically limited conditions is to change the operating temperature significantly and measure the efficiency at different gas velocities as shown by barton et al. (1999) . a large change in removal efficiency at a different temperature indicates kinetically limited as the mass transfer parameters solubility (increases with temperature), diffusion (increases with temperature), and henry's constant (decreases with temperature) are somewhat temperature sensitive, but in general not as significant as the biodegradation parameters. measuring the volumetric removal capacity at different inlet volumetric loadings can be done to identify biological or mass transfer as potential rate-limiting step. when, in different experiments, the empty bed gas contact time is changed at constant substrate loadings, rate-limiting step discrimination could be created, especially when individual reactor sections are monitored as shown for example by paca et al. (2009) . cowger et al. (1992) developed a mathematical expression in order to separate mass transfer and kinetic limitations. they concluded that multiple experiments with various biomass concentrations still must be conducted to yield a definite conclusion of mass transfer-limiting conditions. mathematical expressions, in particular the sensitivity analyses of dimensionless numbers (e.g., damkohler number, thiele modulus, peclet number), can be effective to clarify at least the interplay of mass transfer and biodegradation kinetics. the damkohler number is defined as the ratio of the reaction rate to the mass transfer rate (the sum of advection and diffusion rates). the thiele number is defined as a ratio of the reaction rate to the diffusion rate. the peclet number is the ratio of the rate of advection to the rate of diffusion of compounds in a gas or liquid. sensitivity analyses of dimensionless numbers in biological gas treatment models have been used by different authors (goncalves and govind 2009; aroca et al. 2009 ) to demonstrate successfully whether the operation of the reactor was likely to be mass transfer or kinetically limited. bosma et al. (1996) defined the bioavailability as the inverse of the damkohler number for the case of dissolution controlled biodegradation and proved that the bioavailability number (bn) can be a useful tool to predict threshold concentrations below which no biotransformation is possible in soils slurries and percolation columns. it is important to note that in most situations a reactor is not either mass transfer or kinetically limited, but is a complex combination of both. it could very well be possible that the removal is substrate (mass transfer) limited deep in the biofilm, but kinetically limited near the aqueous/biomass phase. also, as mass transfer and kinetic rates change along the height of reactor, the rate-limiting step might differ along the height of the reactor. finally, as the biomass grows and could accumulate over time, the pore volume of the media can be reduced, reducing the interfacial area between the gas phase and the biofilm. in that case mass transfer limitation can be induced after a long time of operation as adeptly illustrated by popat and deshusses (2010) . in summary, although limited tools exist to determine what the rate-limiting step is in a biological gas treatment system, some authors have demonstrated that they can be used successfully. mass transfer of the target compounds (pollutant and oxygen) from a gas phase into the liquid phase in biological gas treatment systems is often described with the two film theory from lewis and whitman (1924) . this model uses two phases (e.g., gas and liquid) that have different concentrations and are not in equilibrium according to henry's law. only at the gas-liquid interface exists such equilibrium and the target compounds move from or to this interphase with a certain velocity, which is dependent on the type of compound and the properties of the gas and liquid phases. these velocities are defined in mass transfer rate coefficients. the overall mass transfer coefficient (k overall ) is a combination of the different partial intrinsic mass transfer coefficients, often reduced to a mass transfer rate coefficient for the gas phase (k g ), a mass transfer rate coefficient for the liquid phase (k l ), and a mass transfer rate coefficient for the biofilm (k b ) as shown in eq. (1). the mass transfer coefficients are a function of the pollutant physical-chemical properties, the medium properties, the internal reactor characteristics as well as the operating conditions of the reactor system. in suspended reactors (bubble columns, airlift and stirred tanks), eq. (1) continues being valid considering k b as the resistance due to the water film around the cell or flocks of cells. if the resistance to the mass transfer in the gas and the biofilm is negligible (as might be expected in most cases, especially at low pollutant concentrations), the overall volumetric mass transfer rate r (g m −3 s −1 ) from the gas phase to the aqueous phase (where microorganisms are suspended or growing as a biofilm) takes place at a rate that is described (koch 1990) in eq. (2). where d al (m 2 s −1 ), h (dimensionless), and δ film (m) are the gaseous pollutant diffusivity in the liquid, the henry coefficient, and the liquid film thickness, respectively. c g and c l are the pollutant concentrations (g m −3 ) in gas and liquid, respectively. the term k l a (s −1 ) is a volumetric coefficient and consists of all concentration independent factors that determine the mass transfer rate, where k l is the mass transfer coefficient (m s −1 ) and a is the specific interfacial area (m 2 m −3 ) between the gas and liquid phase, regardless of whether a packing is present. when an additional phase (e.g., silicone oil) is added, the specific interfacial area between air and any phase other than water needs to be considered (see also the section on "non-aqueous phase addition" further down this paper). k l and a are often difficult to obtain separate experimentally; however, k l a can be obtained from macroscopic measurements. mass transfer takes place through both diffusion-the random brownian motion of individual compounds in a medium-and by advection, in which compounds are transported by the larger-scale motion of currents in the medium. convection is used to refer to the sum of advective and diffusive transfer. the diffusivity of lowmolecular weight compounds in gas are in generally in the range of 1×10 −5 m 2 s −1 (warneck 1988) and in water in the range of 1×10 −9 m 2 s −1 (harms and bosma 1997) . the diffusivity in biofilms will be far lower as, for example, harms and bosma (1997) reported diffusivity of pollutants in soils and sediments up to 12 orders of magnitude lower than in pure water. from eq. (2), it can be deduced that r can be increased in different ways, for example (a) reducing the liquid film thickness by for instance reducing water flow (biotrickling filters) or increasing mixing (stirred tank bioreactor), (b) increasing the gas-liquid contact area through a support (liquid or solid) or by increasing mixing, and finally, (c) reducing the henry coefficient, which increases the gradient of concentrations (driven force for the mass transfer); this can be done by increasing the affinity between the pollutant and liquid phase, for example by modifying the liquid phase composition. figure 1 shows a schematic representation of mass transfer in a bioreactor for gas treatment applications. as observed in fig. 1 , in a packed or laminar reactor as a biotrickling filter, the pollutant or oxygen can be transferred from the gas flow (f) to the cells through two water films or interfaces, one adhered to the package forming a biofilm, and other with the free water flowing through the package (l). in both interfaces, the pollutant (or oxygen) will be partitioned according to the thermodynamic equilibrium (henry's law). nevertheless, as the equilibrium exists only in the interface (lewis and whitman 1924) , a concentration's gradient between the interface and the liquid bulk leads to a net mass transfer flux (n). as the interface has no volume, the mass transfer accumulation is not possible and equality of fluxes can be established (n g = n l =n b ). figure 1 continues being valid to represent the mass transfer from the gas to liquid in a slurry or suspended bioreactor considering the biofilm on the liquid side as a single cell or flocks of cells dispersed, and the biofilm on the gas side as a single cell or flocks of cells in direct contact with a bubble. a biological gas treatment system contains typically a gas phase, a liquid phase, and a biofilm phase. the overall mass transfer coefficient is therefore a combination of different intrinsic partial mass transfer coefficients of these different phases. below are briefly described some strategies used to estimate or measuring the partial mass transfer coefficients. intrinsic mass transfer coefficients are often calculated by means of empirical correlations, which are a function of the pollutant physical-chemical properties, the internal reactor characteristics (for example the packing material properties), as well as the operating conditions of the reactor system. models describing biofilters or biotrickling filters often use mass transfer coefficients obtained from the empirical correlations developed for gas absorption, stripping or distillation using packing materials (kim and deshusses 2008) . the empirical correlations for packed columns have been summarized by wang et al. (2005) . they compared the main correlations and concluded that the research on modeling the mass transfer prediction for packing materials still remains open as there is often a lack of a scientific basis. a distinction between the correlations to determine the mass transfer coefficient for random packing and structured packing materials was made. more modern (structured) packing materials have been developed that have a high mass transfer capacity at a lower pressure drop, which also comes along with a modified gas and liquid flow behavior. for future improvements of the empirical correlations, they recommended investigation of spatial liquid distribution inside the packing materials and improvement of the experimental setup to obtain more reliable data. also hoffmann et al. (2007) showed discrepancy and inconsistency between different sources in literature data determining mass transfer parameters of packing materials used in absorption columns and concluded that standard procedures for measuring mass transfer of absorption systems are still required. existing correlations are often made for relatively older packing elements. rejl et al. (2009) concluded that, while much data exist, a lot of it has been measured using various different, and often erroneous, methods. this has resulted in the publication of vastly conflicting data and they suggested that the cause of discrepancies is most likely to be found in experimental technique. this is especially true for the sampling procedures (non-elimination of end effects in a packed column), and non-uniform distribution of the gas and liquid phases through the packed column is proposed as the main reasons for these discrepancies, which is an important lesson to future research on mass transfer in biological gas treatment systems. to improve the experimental technique used to determine mass transfer coefficients, it has been proposed (hoffmann et al. 2007 ) that prehumidification to a saturation level be included to avoid both evaporation and condensation, maintain a constant fig. 1 an illustration of mass transfer typical for biological waste gas treatment processes temperature and pressure, prevent wall effects, and guarantee uniform liquid and gas distribution in the experimental setup. as discussed by kim and deshusses (2008) , systematic studies to determine mass transfer coefficients in biological gas treatment reactors are rare and mass transfer estimates are therefore often based on the large body of information obtained for traditional packing materials used in packed columns for gas absorption, stripping or distillation. the process in a biological gas treatment system is different and operating conditions are different, especially in relation to the liquid rate, but also to the gas rate. kim and deshusses (2008) concluded that both gas and liquid mass transfer coefficients for biofilters and biotrickling filter need to be modified, when they are based on empirical correlations that predict mass transfer coefficients for traditional packing columns. also dorado et al. (2009) concluded that there is a need for an accurate method to determine mass transfer parameters specifically in biological gas treatment systems after they showed that existing mathematical correlations for mass transfer coefficients fail to predict their experimentally obtained data. instead of using existing empirical mathematical correlations based on non-standardized experimental setups to predict the mass transfer coefficient, methods to measure the mass transfer coefficients directly on a specific reactor set-up can be used. some of the most common methods are described below. for the liquid-side mass transfer rate the absorption/ desorption of sparingly soluble gases, typically oxygen or carbon dioxide in/from water is generally used to measure k l a. as oxygen has a low solubility in water, there is no significant gas-side resistance. linek et al. (1984) proved experimentally that the values of k l a derived from oxygen absorption and desorption are identical. desorption experiments were used as they were the more simple of the two to carry out. they kept the nitrogen gas flow rate through the packing column low (0.03 ms −1 ), but sufficient to keep the oxygen concentration in the effluent gas lower that 0.2% v/v. this was done to ensure negligible back pressure of oxygen and negligible influence of axial dispersion in the gas phase. the sulfite oxidation method (linek and vacek 1981) , discussed by suresh et al. (2009) , can be used for oxygen mass gas-liquid transfer determination as a reliable, fast, and simple method as it does not require the measurement of dissolved oxygen in the liquid phase. a known amount of sulfite reacts fast in the presence of cobalt into sulfate when oxygen enters the liquid phase. over time the sulfite concentration is determined by iodometric back-titration and the oxygen transfer rate calculated. for the gas-side mass transfer rate, the gas-phase volumetric mass transfer coefficient, k g a, is typically measured using systems whose mass transfer resistance is limited to the gas phase. an instantaneous chemical reaction of a compound in the liquid phase can be used to determine directly the gas-side mass transfer coefficient. different authors (billet and shultes 1999; hoffmann et al. 2007 , linek et al. 2001 ) have used the absorption of so 2 from the gas phase in an aqueous naoh solution, after linek et al. (1995) proved that the absorption rate was independent from the oh concentration across a large concentration range down to 0.01 m. dorado et al. (2009) adopted a method from heymes et al. (2006) to determine both the liquid and gas-side mass transfer coefficients. a packed column acted as a differential adsorption column with water continuously recirculated. a gas flow containing a target compound passes through the column upwards and the compound concentration in the gas inlet, gas outlet and liquid outlet is continuously monitored until the concentration in both the liquid and gas phase remain constant. the method has the advantage that it makes it is possible to use the target compound to be treated in the gas treatment system. they included suggestions from hoffmann et al. (2007) to minimize most common errors found in mass transfer studies. dorado et al. (2009) pointed out that in typically used biological gas treatment systems (biofilter and biotrickling filters), the gas phase resistance should not be neglected at high gas contact times (low gas velocities), probably because diffusion in the gas phase is the dominant process rather than advection. cowger et al. (1992) developed a mathematical expression in order to separate mass transfer and kinetic limitations and elegantly showed that when multiple experiments with various cell concentrations are conducted under mass transfer-limiting conditions, the overall mass transfer coefficient can be obtained from removal efficiency measurements at different gas flows. once the volumetric mass transfer coefficient has been determined, the overall volumetric mass transfer rate r in eq. (2) can be calculated for the maximum concentration gradient, considering that under mass transfer-limiting conditions the dissolved concentration of the target compound is zero as it is degraded immediately by microorganisms. the intrinsic mass transfer coefficient is a function of the pollutant physical-chemical properties, the medium properties (e.g., viscosity, salt and organic content), the internal reactor characteristics (e.g., gas and liquid flow behavior, surface area and wettability of the packing material), as well as the operating conditions (e.g., gas velocity, liquid velocity, ph, and temperature). some of these parameters and their relevance to biological gas treatment systems are discussed below. the overall mass transfer coefficient k l a (s −1 ) is directly related to the effective interfacial area (m 2 m −3 ). the effective interfacial area is often different from the specific surface area (a) of packing material, as part of the packing surface might not be wetted or could be located in a dead zone not being effective for mass transfer. water droplets and swirls on the other hand could increase the effective interfacial area. the effective interfacial area depends on the operation conditions of the system. physical methods such as electro-resistivity, light transmission, and reflection techniques can be used, but usually the effective interfacial area is determined by mass transfer measurements in the presence of a fast chemical reaction as proposed by joosten and danckwerts (1973) . this method uses the absorption of relatively low concentrations (< 1% v/v) carbon dioxideenriched air in 1 m naoh solution. rejl et al. (2009) showed that this method can only be used when the gas velocity is >0.5 ms −1 , otherwise the gas phase resistance becomes too large. although the required gas velocity is in the range (0.3-3 ms −1 ) typically found in packed columns used for absorption (e.g., chemical scrubbers) or distillation and evaporation, it is too high for most biological gas treatment systems, which normally operate in the range 0.02-0.4 ms −1 . therefore, this standard method might be less suitable to determine the exact effective interfacial area in a biological gas treatment system. packing materials have specific surface areas and not all parts of the surface may contribute to mass transfer as, for example, not all parts are wetted at all times. the wetted specific surface area is a percentage of the total specific surface area of the packing material, also sometimes expressed as wettability factor, and is dependent on factors such as gas and liquid flow. this wettability is critical for the overall performance for packed columns used for absorption (e.g., chemical scrubbers) or distillation and evaporation, which might not necessarily be true for all biological gas treatment systems. the catalytic reaction in a chemical scrubber normally takes place in the liquid phase, while the catalytic reaction in most common used biological gas treatment systems (biofilter and biotrickling filter) takes place not so much in the liquid phase but for the majority in the biofilm, even in biotrickling filters with continuous recirculation (cox et al. 2000) . also due to biological growth on the packing material the surface becomes hydrophilic and therefore wettability is much higher than the bare surface properties. direct gas-biofilm mass transfer is preferred when the catalytic reaction takes place in the biofilm. in a biological system, the liquid is required to prevent drying out of the biofilm and is a transport medium for the supply of nutrients and sometimes the removal of degradation products (e.g., sulfuric acid). transport of water over the biofilm is necessary to maintain a high water activity, but can actually form an extra barrier for mass transfer as illustrated by popat and deshusses (2010) among others where the elimination capacity in their biotrickling filter experiment was the highest when recirculation of the liquid was temporarily stopped. another important factor is the air distribution, which is most of the time not taken into consideration when modeling biofiltration. most models presume that air flow within the reactor is plug flow (devinney and ramesh 2005) , which might be a too simplified assumption. prenafeta-boldu et al. (2008) showed the importance of the presence of stagnant air zones in different packing materials and demonstrated that increasing airflows reduces stagnant air zones and can consequently reduces potential diffusion limitations. many biological gas treatment models are based on the equilibrium according to henry's law at the gasliquid interface. the presence of biomass can have a substantial effect on this equilibrium as illustrated by davison et al. (2000) and barton et al. (2003) and further discussed by barton et al. (2008) . high biomass or organic levels of more than 15 g dry weight per liter can be found in biofilms present in biological gas treatment systems. the overall mass transfer is directly related to the henry coefficient and measurements of henry coefficients and maximum solubility in aqua-biomass mixtures are necessary to compensate for the additional organic constituents. the henry coefficient of, for example, toluene decreased by a factor of 30 compared to pure water when the biomass content was 30 g dry weight per liter and its maximum solubility increased by a factor 4 compared to pure water when biomass content was 0.25 g dry weight per ml. at 100 g biomass per liter, the henry's value for trichloroethene was only about 2% of that in pure water and even at relatively low biomass levels, such as 10 g per liter, the partitioning constant was less than 10% of that in pure water. in the absence of these experimental data, barton et al. (2008) recommended use of octanol-water partitioning constants rather than the gas-water partitioning constants. although the authors showed that the use of octanol-water partitioning constants is not a reliable predictor, they illustrated that the effect of biomass on the henry coefficient (trend and order of magnitude) is described correctly. miller and allen (2005) discussed the transport of hydrophobic compounds through biofilms and concluded that a biologically mediated transformation of a hydrophobic compound into a more soluble, less volatile by-product that then can penetrate deeper into the biofilm can take place. not only the presence of biomass, but also the presence of extracellular metabolites such as biosurfactants could, for example, affect the overall mass transfer (painmanakul et al. 2005) . basically, we can divide the bioreactors for gas treatment operations in two groups: turbulent and laminar contactors. in turbulent contactors (stirred tanks, bubble columns, or airlift reactors), the energy supplied to the system is used to break bubbles and to reduce the liquid film (increasing a and reducing δ film in eq. 2, respectively); nevertheless, above an optimal value of power input most of the energy is dissipated in the liquid through eddies that do not contribute to the mass transfer (kreutzer et al. 2006) increasing the operation costs of process. in laminar contactors (biofilters and biotrickling filters), a structure (media) is used to provide the gas/ liquid contact area, the power consumption in these systems can be 1 or 2 orders of magnitude lower than in turbulent contactors (rocha-rios et al. 2010) , and this explains why for commercial applications, biofilters, and biotrickling filters continue being the most used technologies. nevertheless, the main limitation of laminar contactors is related to mass transfer limitations of poorly water soluble compounds. typical removal efficiencies of compounds as acetone, ethanol or toluene are higher than 90% (adler 2001) where the removal efficiencies for poorly soluble compounds as methane are typically lower than 70% (rocha-rios et al. 2010) and requiring large gas contact times in the reactor. unlike the turbulent systems where the suspensions gas/ liquid and cells/liquid can be assumed perfectly mixed and the mass transfer rate can be expect to be similar at all points of the reactor, in laminar contactors (biotrickling filters and biofilters) operational problems are often caused by heterogeneities generating gradients in pollutant, water content, and biomass concentrations as well as gradients in nutrient concentration and ph through the package (ramirez-lopez et al. 2000) . moreover, due to the presence of heterogeneities throughout the reactor, especially the water content causes differences in the support's compaction resulting in flow channelization and possible anaerobic regions formation (cardenas-gonzalez et al. 1999) . to avoid compaction problems in random laminar contactors, new supports covering a wide variety of specific surface areas and porosity have been developed reducing the pressure drop problems in these systems. an interesting laminar contactor that prevents the formation of heterogeneities with minimal power consumption is the capillary reactor when operated under taylor flow and is discussed later in this paper. as technologies for biological gas treatment are applied mostly at relatively low gas concentrations, we estimate that they are nearly always subject to at least partial mass transfer limitation. mass transfer limitation of either substrate or oxygen can occur near the aqueous/biomass phase, deep in the biofilm, or just near the exit of the system due to the low partial pressure of the target compound. biotechnology for gas treatment is accepted as an economical and reliable air pollution control technology for treating gases contaminated mainly with relatively low concentrations. the extension of its application field is limited in many cases by the mass transfer of especially hydrophobic target compounds. below we briefly review some of the strategies which we have shown to be technical feasible to overcome mass transfer limitation. fungi have the advantage that their aerial mycelia form a larger surface area (see eq. 2) in the gas phase than bacterial biofilms, which is suggested to facilitate the uptake of hydrophobic volatile compounds (cox 1995) . wosten et al. (1999) and wosten (2001) illustrated the role of hydrophobins in fungal growth, how they influence hydrophobicity of the aerial mycelia of fungi and showed that the presence of water hinders the development of the aerial mycelia. filamentous fungi secrete hydrophobins at hydrophobic-hydrophilic interfaces such as gas-water to form an amphipathic coating that lowers the surface tension, which enables hyphae to breach the water-gas interface. this mechanism can be used by fungi for direct pollutant consumption from the gas phase avoiding the mass transfer resistance in the liquid. arriaga and revah (2009) determined the partition coefficient between hexane and (wet or dried) fungal samples (perlite + fusarium solani) and between hexane and dried bacterial samples (perlite + consortium). the results indicated that partition coefficient of hexane was nearly an order of magnitude lower for fungal (more absorption) than bacteria films, and it was lower for dry fungal samples than for the wet samples. this proves also that direct gas-biofilm mass transfer is preferred, minimizing the gas-liquid-biofilm path for the transfer of the target compound. vergara-fernandez et al. (2006) illustrated that the surface hydrophobicity of the fungi mycelia can change over time and increase consistently with more hydrophobic substrates. besides relatively higher mass transfer rates of hydrophobic compounds in fungal biofilters than in bacterial biofilters, another advantage is that fungi are more resistant to drying out and acidification. fungi biofilters have shown to be significantly more robust to poor moisture control in the biofilter (cox 1995; kraakman et al. 1997; groenestijn et al. 2001) . this makes it possible to operate a fungal biofilter under relatively dry condition reducing the liquid film thickness (δ film in eq. 2). different authors (groenestijn et al. 2001; arriaga and revah 2005a, b; spigno et al. 2003; jin et al. 2007) showed relatively high removal capacities of hydrophobic compounds in fungal biofilter and illustrated the improved robustness during typical upsets that can be experienced in an industrial application like interruption of water, gas, and nutrient supply. table 1 presents different studies where a fungus, as a dominant organism, was used for pollutant degradation. high conversion rates come with increased biomass growth in biofilters and high pressure losses are reached sooner with filamentous fungi than with non-filamentous microorganisms, eventually causing clogging and channeling problems in the biofilter. innovative solutions, such as the addition of higher organisms, are explored and described by groenestijn et al. (2001) and woertz et al. (2002a, b) . this "grazing" concept showed that the use of higher organisms (mites) in a fungal biofilter allowed maintenance of a low pressure drop and minimize power consumption. the fungi grow partially in the pores of the media (perlite) protected against predation, while the mites grow by "grazing" at a relatively low biomass yield and are partly washed out as a result of the discontinuous irrigation. simultaneously, the performance of the fungal biofilter was significantly higher compared to the control fungal biofilter without mites. the addition of a non-aqueous phase liquid (napl) such as for instance an organic solvent can overcome design and operational limitations of biological systems. interesting advances have been made over the last 15 years in different biological systems that make use of an organic phase as transfer vectors, despite the lack of knowledge of the exact mechanisms (déziel et al. 1999; quijano et al. 2009a ). the so-called two-phase partitioning bioreactors make use of an extra phase (solvent) to enhance the productivity or facilitate the downstream processing from bioprocesses. the concept is applied either to control the delivery of a (sometimes toxic) substrate dissolved in the non-aqueous phase or to continuously extract a bio-product (ratledge 1977; wubbolts et al. 1996) . this technology has now been researched for the treatment of poorly water soluble gaseous compounds (yeom and daugulis 2001; davison and daugulis 2003; arriaga et al. 2006) . the non-aqueous phase (for example a large branched alkane, silicone oil or a plastic polymer), which is selected as immiscible, nonvolatile, non-toxic, non-biodegradable, and with high affinity by the target compound (quijano et al. 2010a; hernandez et al. 2010; rocha-rios et al. 2011a) , is added to the aqueous phase with the aim of improving the transfer of hydrophobic substrates from the gas phase, buffering fluctuations in the gaseous concentrations or reducing the toxicity for the microorganisms. the review by muñoz et al. (2007) shows that the main transfer mechanisms are based on the hypothesis that the substrate uptake takes place in the aqueous phase. resistance of the diffusion through the liquid boundary layer is counteracted both by the increase in the interfacial area for mass transfer and the enhanced accumulation of the target compound in the organic phase, which acts as a reservoir. they also noted that various authors have observed bacterial adhesion at the non-aqueous/aqueous interface (rosenberg 1991; mcleod and daugulis 2005) and have suggested that direct pollutant and oxygen uptake from the non-aqueous phase improved the overall removal efficiency of the process. it is also known that many microorganisms are also capable of producing biosurfactants that can solubilize the hydrophobic compounds and possibly improve uptake (hamme et al. 2003; mcleod and daugulis 2005) . with the addition of a solvent, the overall change in volumetric mass transfer coefficient (k l a) depends on the relative magnitudes of effects on a new resistance (organic phase) added to the overall system, which is usually higher than that of water due to a higher viscosity of the napl, and the different interfacial contact areas that can be present (gas-water, gas-oil, oil-water, cell-gas, cell-water, cell-oil). table 2 shows different studies where an organic phase was added to increase the pollutant or oxygen transfer from the gas to the liquid. in turbulent reactors, for the organic phase addition to be effective, the increase in the different interfacial contact areas must overcome the general increase in the mass transfer resistance by the organic phase addition (clarke and correia 2008; quijano et al. 2010b) . it has been shown in stirred tank reactors that silicone oil drops can increase arriaga and revah (2009) the gas-water interfacial contact area through two effects, first, colliding with the gas bubbles and breaking them (galindo et al. 2000; quijano et al. 2010b) , and second, by a reduction in the gas-water surface tension (quijano et al. 2010b ). the first effect is increased by the stirring rate (power consumption) in the system as demonstrated by rocha-rios et al. (2010) who observed no effect of silicone oil addition (5 and 10% v/v) on methane degradation at 200 rpm, while a positive effect was observed at 500 and 800 rpm. obtaining a mathematical model to describe the effect of a napl in these systems is still a challenge. the first attempt to model the degradation of a gaseous pollutant (hexane) in a two-phase partition bioreactor was developed by bordel et al. (2010) considering that all hexane degradation takes place in the aqueous phase. this assumption cannot be valid for more hydrophobic compounds like methane as han et al. (2009) reported that most of methylosinus trichosporium cells using methane as the sole carbon source were growing attached to the paraffin oil drops used as a mass transfer vector. rocha-rios et al. (2009 proposed that a possible direct uptake of methane could be important to explain the increase in the elimination capacities observed. contrasting results were reported by quijano et al. (2009b) using silicone oil as oxygen transfer vector, who found through sulfite oxidation determinations, that 90% of the oxygen transfer was airwater and just 10% air-oil-water. therefore, more studies are required to clarify this particular finding. moreover, the empirical correlations that are used to estimate k l a properly in turbulent one-liquid phase systems, may be misleading in two-phase partition systems as soon as the viscosity exceeds ten times the 1 mpas viscosity of pure water (delaloye et al. 1991; rocha-rios et al. 2010 ). although dorado et al. (2009) showed that at low gas velocities the mass transfer can be determined by the gas phase, the mass transfer is usually limited by the liquid-side resistance. experiments by heymes et al. (2006) showed that the k l a of a system using viscous liquids depends on the liquid velocity but also on the gas velocity. this behavior has also been observed by the few authors who have used viscous fluids in their experiments and is contrary to all the authors who have worked on lowviscosity fluids. the physical characterization of the liquid phase plays an important role in hydrodynamics and mass transfer kinetics. gomez-diaz and navaza (2003) showed, for example, that the presence of polymers (carboxymethyl cellulose) influences the viscosity and thereby the mass transfer. it is therefore clear that the influence of viscosity on the mass transfer phenomenon is considerable and should be taken into account at the addition of a solvent. the stability of a non-aqueous solvent under long-term operation is, in general, unclear and its application could be limited by the fact that the solvent can be slightly volatile. to overcome this, highly stable solid polymers based on copolymers of polyurethane, vinyl acetate, and ethylene (e.g., kraton, elvax, desmopan) as the non-aqueous phase has been proposed as well as hydrophobic packing materials (montes et al. 2011) . although slower to respond to sudden changes in pollutant concentration compared with the liquid-liquid systems (boudreau and daugulis 2006) , the overall biological system can be more stable and possibly more cost effective. in laminar reactors, different heterogeneities are produced in the package of biofilters and biotrickling filters during the operation (biomass, humidity, air canalization, etc.; cardenas-gonzalez et al. 1999) . when an organic phase (like silicone oil) is added, its distribution through the package is affected by these heterogeneities generating zones in the package with higher oil content (this effect is enhanced by the higher viscosity of the oil than water). this irregular oil distribution, which is a consequence of mixing lack in the package, will be increased as a function of the operation time, unless a periodic interruption of the bioreactor's operation is made to mix the package. unlike the turbulent reactors where the organic phase addition has increased generally the removal efficiency of hydrophobic pollutants (table 2) , inconclusive results have been obtained in laminar contactors for the same pollutants at similar pollutant loadings. in the laminar reactors shown in table 2 , an increase in elimination capacity was reported with the organic phase addition; however, in most of these studies a higher pollutant load was fed in the two-phase systems, producing similar removal efficiencies that in control systems (without organic phase addition). fazaelipoor and shojaosadati (2002) observed no effect of silicone oil addition (20% v/v) on biofiltration of a mixture of hydrophobic compounds with hexane as the principal component (80%) at low inlet concentrations (1 gm −3 ), but when the inlet concentration was increased at 3 gm −3 , an improvement of 10% in the removal efficiency was obtained. a strategy to avoid the addition of an organic phase in laminar contactors is selecting a packing material with similar properties (fundamentally high affinity by the pollutant and oxygen) (montes et al. 2011) . another strategy to avoid the formation of heterogeneities caused by a random distribution of the packed particles, is changing to a structured package (capillary bioreactor) as proposed by kreutzer et al. (2005b and , ebrahimi et al. (2005a) and rocha-rios et al. (2011b) as discussed further up in this paper. often a non-aqueous phase liquid like silicone oil does not mix well with water due to differences in hydrophobicity and density, which induces partitioning between the two phases. if not controlled, phase separation could very well induce a reduction of the overall biodegradation efficiency. stable emulsions are required and might require additional mixing. we have seen that the stability of a silicone oil and water emulsion improves over time in the presence of microorganisms (rocha-rios et al. 2011b) . the non-aqueous phase should be selected based on its cost, enhanced partitioning properties towards the target compound(s), immiscibility, viscosity, volatility, safety, nontoxicity, and non-biodegradability. although data on costs, immiscibility, viscosity, toxicity, volatility, safety, and biodegradability are available for different solvents and solid polymers (quijano et al. 2010a; hernandez et al. 2010; rocha-rios et al. 2011a) , sufficient data are at this moment not yet available to predict the partitioning properties of non-aqueous phase towards many target compounds. the traditionally used biological gas treatment methods (biofilters and biotrickling filters) can be called laminar contactors. laminar flow occurs when a gas or liquid flows in parallel layers, with minimal disruption between the layers. laminar flow is a flow regime characterized by high diffusion and low advection and is the opposite of turbulent flow. the mass transfer rate through a water film by diffusion is relatively slow when compared to diffusion through gas (in general by a factor of approximately 10,000). therefore improved convection by advection (for example through mixing) will improve mass transfer through a water film. considerable effort has been expended in the search of less energy-intensive reactors to further enhance mass transfer rate. capillary reactors combine good mass transfer with low pressure drop, two important factors affecting cost effectiveness for many industrial applications. capillary reactors are structures of parallel straight microchannels (capillary channels) separated by a thin wall. the hydrodynamics of gas-liquid flow in capillary channels have been studied within the context of chemical reaction engineering (nijhuis et al. 2002; kreutzer et al. 2005b; shao et al. 2010) . a capillary reactor is a reactor where the capillary forces, created by the capillary channel, become dominant over other forces as such gravity and viscosity. for air-water contractors, this means a capillary channel with a diameter around 5 mm or smaller ). the preferred flow patron, called segmented flow or taylor flow, is a bubble train of alternating liquid slugs and air bubbles with gas and liquid flowing downwards or upwards co-currently. although the airflow seems to be laminar, the internal liquid circulation increases the mass transfer from the gas phase to the liquid phase where a plug flow (no macromixing and axial dispersion) is combined with good mass transfer (local mixing) and low pressure drop . a bioreactor using a monolith support operated under taylor flow conditions is an example of a capillary bioreactor. the rate of transport of the compounds through a medium is characterized by resistance to the medium. input of energy can overcome resistance, such as, for example, mixing in bubble-tank bioreactor to break up air bubbles and thereby increasing the interfacial surface area of the gas bubble with the liquid. the physical input of energy in a system is always limited by the equipment required for the energy input, which will operate with a specific optimal efficiency dependent on its operating conditions. the specific efficiency for example of a mixer is typically around 60-70% when operated under optimal conditions. increased energy consumption results in increased cost of operation, which should be minimized for industrial applications. the mass transfer using taylor flow is relatively energy efficient, mainly because no energy is required to maintain the small gas bubble size. figure 2 shows the mass transfer rate versus energy input for biotrickling filtration and capillary reactors. the data for capillary reactors were obtained from , who illustrated, with an order of magnitude analysis, that the relation between mass transfer rate and the power input per reactor volume for a capillary reactor can be expressed as k l a % 0:1 â p=v ð þ 0:25 , with p the power input (watt) and v the reactor volume (m 3 ). the data for the biotrickling filter were obtained from kim and deshusses (2008) , who were the first to conduct a systematic study to actually measure mass transfer rates in the most common used biotechnologies for gas treatment (biofiltration and biotrickling filtration). capillary reactors are becoming increasingly significant as multiphase reactors, considering the advantages that they offer, in comparison with conventionally used trickle beds or biofilters for a host of processes (liu et al. 2005) . these advantages, which include low pressure drop, high gasliquid mass transfer rates, and minimum axial dispersion (plug flow), stem from the uniquely structured multichannel configuration of capillary channels. some studies have shown that the use of capillary reactors, in lieu of trickle beds, results in higher productivities and a very significant reduction in reactor size for specified chemical processes (nijhuis et al. 2002; stankiewicz 2001) . ebrahimi et al. (2005a, b) studied the biomass growth in capillary (monolithic) bioreactors and concluded that biofilm formation can be minimized by a proper choice of operation and that periodic biomass removal is relatively simple by ringing with water at moderate pressures. rocha-rios et al. (2011b) illustrated that the addition of a nonaqueous phase (silicone oil) in a capillary bioreactor removing methane is beneficial, which shows the potential of capillary bioreactor for the treatment of compounds with poor water solubility. understanding the mass transfer behavior in bioreactors for gas treatment is highly relevant to obtain improved modeling tools and more advanced reactor operations. different aspects have been discussed and it shows that work is still needed to fully understand the phenomena of mass transfer in a bioreactor for gas treatment. studies to determine mass transfer coefficients in biological gas treatment reactors are rare and mass transfer estimates are therefore often derived from studies on packing materials in systems (e.g., absorption columns) with different process conditions. the influence of biomass on the partitioning coefficient of the target compound in a gas-liquid and gasbiofilm interface in biological gas treatment processes also needs more detailed study. some of the strategies that have shown to be technical feasible to overcome mass transfer limitation have been reviewed. although the mechanisms of mass transfer enhancement are not fully elucidated and different technical challenges need to be resolved before they can be used at full-scale, several promising strategies to improve mass transfer while minimizing power consumption mark the future trend. in random package contactors, these strategies include the use of inorganic supports with high specific surface areas and high porosities, the action of fungi and the addition of a liquid organic phase. nevertheless, the addition of an organic phase, more viscous than water, may increase the formation of heterogeneities through the bed affecting the pollutant transfer rate. this could explain why different authors have reported either positive or negative effects of the organic phase on mass transfer in these systems. an interesting alternative is the absorption of 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reactor techniques for oxygen transfer measurement in bioreactors: a review phase partition of gaseous hexane and surface hydrophobicity of fusarium solani when grown in liquid and solid media with hexanol and hexane review of mass-transfer correlations for packed columns a fungal vapor-phase bioreactor for the removal of nitrous oxide from waste gas streams dynamic bioreactor operation: effects of packing material and mite predation on toluene removal from off-gas mite growth on fungus under various environmental conditions and its potential application in biofilters hydrophobins: multipurpose proteins how fungus escapes the water to grow into the air biosynthesis of synthons in two-liquid-phase media development of a novel bioreactor system for treatment of gaseous benzene acknowledgment the financial support for this work was given by the netherlands organisation for scientific research (nwo), bioway international b.v., and the technical university delft, the netherlands under nwo-casimir project 018.002.019.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 